Methods and Compositions for Inhibiting Diseases of the Central Nervous System

ABSTRACT

Methods and compositions for treating central nervous system diseases and disorders are disclosed.

This application claims priority under 35 U.S.C. §119(e) to U.S.Provisional Patent Application No. 61/134,350, filed on Jul. 9, 2008 andto U.S. Provisional Patent Application No. 61/208,090, filed on Feb. 20,2009. The foregoing applications are incorporated by reference herein.

This invention was made with government support under 5R01NS034239-14and P01 NS43986-06 awarded by the National Institutes of NuerologicalDisorders and Stroke, National Institutes of Health. The government hascertain rights in the invention.

FIELD OF THE INVENTION

The present invention relates to the fields of central nervous systemdisorders. More specifically, the invention provides compositions andmethods for the treatment of central nervous disorders, particularlyParkinson's Disease.

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thespecification in order to describe the state of the art to which thisinvention pertains. Each of these citations is incorporated herein byreference as though set forth in full.

Parkinson's disease (PD) is a common progressive neurodegenerativedisease clinically characterized by resting tremor, muscle rigidity,bradykinesia, and postural instability (Dauer et al. (2003) Neuron39:889-909). PD is sporadic and of unknown cause although host genetics,environmental cues, aging, impaired energy metabolism and oxidativestress are linked to disease onset and progression (Klockgether, T.(2004) Cell Tissue Res., 318:115-120). Pathologically, PD ischaracterized by degeneration of dopaminergic cell bodies in thesubstantia nigra pars compacta (SNpc) and their associated caudateprojections (Dauer et al. (2003) Neuron 39:889-909). Nonetheless, thepathological hallmark of PD is cytoplasmic inclusions of fibrillar,misfolded proteins called Lewy bodies composed principally ofα-synuclein (α-Syn) (Spillantini et al. (1997) Nature, 388: 839-840).

α-Syn is a 140-amino acid (aa), natively unfolded, soluble protein thatis localized in the pre-synaptic terminals of neurons of the centralnervous system (CNS), where it interacts with and may regulate synapticvesicles (Spillantini et al. (1997) Nature 388: 839-840; Sidhu et al.(2004) FASEB J., 18:637-647; Paxinou et al. (2001) J. Neurosci.,21:8053-8061; Weinreb et al. (1996) Biochemistry 35:13709-13715; Eliezeret al. (2001) J. Mol. Biol., 307:1061-1073; Uversky et al. (2000)Proteins 41:415-427). Three missense mutations (A53T, A30P and E46K) inthe gene encoding α-Syn are linked to dominantly inherited PD (Kruger etal. (1998) Nat. Genet., 18:106-108; Polymeropoulos, et al. (1997)Science, 276:2045-2047; Zarranz et al. (2004) Ann. Neurol., 55:164-173).Moreover, multiplication of the wild-type (WT) gene has also been linkedto PD, suggesting that the level of α-Syn is an important pathogenicfactor (Chartier-Harlin et al. (2004) Lancet 364:1167-1169; Singleton etal. (2003) Science 302:841). Such familial cases are rare and insporadic PD, there is no genetic aberration of α-Syn. However, it hasbeen proposed that post-translational modifications such as nitrationenhances WT α-Syn propensity to aggregate (Hodara et al. (2004) J. Biol.Chem., 279:47746-47753; Uversky et al. (2001) J. Biol. Chem.,276:10737-10744; Uversky et al. (2005) Brain Res. Mol. Brain. Res.,134:84-102; Yamin et al. (2003) FEBS Lett., 542:147-152). Oxidized andaggregated α-Syn, when released from dying neurons, may stimulatescavenger receptors on microglia resulting in their sustained activationand dopaminergic neurodegeneration (Wersinger et al. (2006) Curr. Med.Chem., 13: 591-602; Zhang et al. (2005) FASEB J., 19:533-542; Croisieret al. (2005) J. Neuroinflammation 2:14). Moreover, activated microgliagenerate nitric oxide and superoxide that rapidly react to formperoxynitrite which can then traverse cell membranes resulting in3-nitrotyrosine (NT) formation, DNA damage, mitochondrial inhibition, orlipid peroxidation (Dringen, R. (2005) Antioxid. Redox. Signal7:1223-1233; Ischiropoulos, et al. (2003) J. Clin. Invest.,111:163-169).

SUMMARY OF THE INVENTION

In accordance with the instant invention methods of treating a centralnervous system disease or disorder in a patient in need thereof areprovided. In a particular embodiment, the central nervous system diseaseor disorder is characterized by the presence of at least one abnormalprotein. In yet another embodiment, the methods comprise administeringto the patient a) at least one immunogen capable of inducing a humoralimmune response against the abnormal protein, and b) at least oneadjuvant that stimulates regulatory T cells.

In accordance with another aspect, compositions are provided forperforming the methods of the instant invention. In a particularembodiment, the composition comprises a) at least one immunogen capableof inducing a humoral immune response against at least one abnormalprotein of a central nervous system disease or disorder, and b) at leastone adjuvant that stimulates regulatory T cells. In one embodiment, thecomposition further comprises at least one pharmaceutically acceptablecarrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F demonstrate drainage of N-α-Syn and MBP to CLN withmacrophage activation and production of α-Syn serum antibodies afterMPTP intoxication. FIG. 1A provides a Western blot of tissue homogenatesfrom VMB and CLN of mice 20 hours following treatment with PBS or MPTP,probed with antibodies to α-Syn. FIG. 1B provides results of an N-α/βSyn IP with (clone nSyn12 antibodies) against CLN homogenates from PBSor MPTP-treated mice. Immunoprecipitates were fractionated on a 16%polyacrylamide gel and the gel stained with SYPRO® Red or blotted. TheWestern blot was probed with anti-α-Syn. Proteins recovered from in-geldigestion of 12-18 kD fragments from anti-N-α/β Syn of CLNimmunoprecipitates were identified by LC-MS/MS. The sequence coverage bypeptides identified by LC-MS/MS from the CLN of MPTP-treated mice ishighlighted in yellow within the primary amino acid (aa) sequence offull-length mouse α-Syn (FIG. 1C; SEQ ID NO: 1). FIG. 1D providesWestern blots of lymph node homogenates (Cervical, Axillary, andInquinal) from mice treated with PBS or MPTP. Blots were probed withantibodies to nitrotyrosine (NT) or anti-myelin basic protein (MBP).FIG. 1E provides results from a flow cytometric analysis of CD11b andI-Ab expression in cells from CLN, show an increased number ofCD11b⁺I-A⁺ cells 24 hours after MPTP treatment compared to PBSadministered animals (n=3 mice/group). FIG. 1F demonstrates antibodiesagainst α-Syn and N-α-Syn in sera of B6 WT mice on day 21 following MPTPintoxication (n=8) or PBS control treatment (n=5) as determined byanti-α-Syn specific ELISA. Sera from MPTP treated group containedsignificantly higher IgG antibodies directed against 4YSyn (p=0.021) andN-4YSyn (p=0.016) compared to PBS treated control sera. Comparisons ofmean IgG concentrations±SEM was by Student's t test.

FIGS. 2A-2C demonstrate nigral degeneration following MPTP-intoxicationin B6 SCID mice before and after lymphoid cell reconstitution. FIG. 2Aprovides photomicrographs of TH-immunostained SN (left panels) andCD3-immunostained spleen sections (right panels) from B6 (WT), SCID, andreconstituted SCID (RCS-SCID) mice treated with PBS or MPTP and obtainedon day 21 post-MPTP intoxication. Immunostaining for expression of CD3in spleens show normal distributions of CD3⁺ T cells in B6 WT andRCS-SCID mice treated with PBS or MPTP. Note the absence of CD3⁺ T cellsin spleens from SCID MPTP mice. FIG. 2B provides the quantification ofTH+ neurons in the SN of B6 WT, SCID, or reconstituted (RCS) SCID micetreated with PBS or MPTP. Values represent mean number of TH+neurons±SEM for 5-9 mice per group. ^(abcdefg)pair-wise comparisons byBonferroni post-hoc test: ^(acd)p<0.0001, ^(bef)p<0.001, ^(g)p<0.05.FIG. 2C provides coronal VMB sections of MPTP intoxicated B6 micereacted with antibodies against CD3, CD4 and CD8 show positiveimmunostaining of cells with small, round lymphocytic morphology(magnification=400×).

FIGS. 3A-3D provide the characterization of purified and nitratedrecombinant 4YSyn. FIG. 3A provides the primary aa sequence ofHis-tagged 4YSyn peptide (SEQ ID NO: 2). The His-Tag sequence ishighlighted. The sequence of 4YSyn (Syn100-140) is shown underlined with4 Tyr residues highlighted as potent sites for nitration. Trypsincleavage sites at Arg (arrowhead) and Lys (arrow) are shown. FIG. 3Bshows purified 4YSyn (lane 1) and N-4YSyn following nitration withperoxynitrite (lane 2) fractionated on a 10-20% polyacrylamide gel andvisualized using silver stain. Covalently cross-linked oligomers areindicated by arrowheads. FIG. 3C provides Western blot confirmation ofpurified 4YSyn and its associated NT modifications followingperoxynitrite treatment. FIG. 3D provides MALDI-TOF spectra of purified4YSyn (top panel), N-4YSyn (middle panel), and 4YSyn after trypticdigest (lower panel).

FIGS. 4A-4B provide the experimental protocol for adoptive transfer andlymphocyte proliferation assessment of donor SPC in B10.BR mice. In FIG.4A, B10.BR (H-2K) mice were immunized with PBS, 50 μg 4YSyn, or 50 μgN-4YSyn emulsified in CFA. Mice were boosted 14 days later with PBS ortheir respective antigens in IFA. After 5 days, donor mice weresacrificed and single cell suspensions were prepared from the draininginguinal lymph nodes and spleen, and T cells were enriched by negativeselection. Twelve hours after the final MPTP injection, 5×10⁷ donorimmune SPC or 2.5×10⁷ T cells were adoptively transferred toMPTP-treated recipient mice. SPC were evaluated for antigen specificityprior to adoptive transfer by lymphocyte proliferation assays. SN ofrecipients were evaluated after 28 days of MPTP treatment for migrationof T cells, survival of dopaminergic neurons, and reactive microglia. InFIG. 4B, SPC were tested for antigen specific proliferation by culturingin the presence of media alone or media containing 3 μg/ml of immunizingantigens for 5 days and using standard 3H-thymidine incorporationassays.

FIGS. 5A and 5B demonstrate that adoptive transfer of SPC and purified Tcells from N-4YSyn vaccinated B10.BR donors leads to infiltration of Tcells in the SNpc of MPTP mice on day 2. FIG. 5A demonstrates thefrequency of CD3⁺ T cells and CD19+ B cells before and after enrichmentof T cells. Population of enriched T-cells was 94% CD3⁺ prior toadoptive transfer to B10.BR mice. FIG. 5B demonstrates that sectionsthroughout the SNpc were immunostained for CD3 and counterstained withthionin. Clusters of CD3⁺ cells are observed within the SNpc(arrowheads) as seen at 100× magnification (left). Magnification (600×)of boxed area (left panel) is shown (right panel). CD3⁺ cells are smalland round exhibiting lymphocyte morphology.

FIG. 6 demonstrates that SPC from N-4YSyn immunized B10.BR miceexacerbate MPTP-induced dopaminergic neurodegeneration and inducemicroglial responses in the SNpc. Photomicrographs from VMB sectionsstained with Fluoro-Jade C (left and middle panels) and Mac-1 antibody(right panels). PBS controls (PBS/none) exhibit an absence ofFluoro-Jade C stained dead neurons on days 2 and 7, and only faint Mac-1immunoreactivity on day 4 post-treatment. In MPTP-treated mice thatreceived SPC from PBS/adjuvant treated donors (MPTP/PBS), Fluoro-Jade Cstained neurons are evident at day 2, but not detectable by day 7.MPTP-treated mice that received SPC from 4YSyn immunized donors(MPTP/4YSyn), also exhibits dead fluorescent neurons by day 2 comparableto the MPTP/PBS control group, and only rare degenerating neurons arevisible by day 7. Mac-1 immunoreactivity in those mice is comparablyresolved to levels seen in MPTP/PBS control group. SPC transfers fromN-4YSyn immunized donors to MPTP-treated mice (MPTP/N-4YSyn) induced arobust and prolonged microglial response, conspicuously enhanced whencompared to MPTP/PBS-treated controls, with concomitant neuronal deathstill evident by Fluoro-Jade C staining at day 7.

FIGS. 7A and 7B show N-4YSyn immunization with adjuvant exacerbatesdopaminergic neuronal cell loss in B10.BR mice. All panels of FIG. 7Ashow TH-positive neurons in the SN from mice treated with: [top row, Lto R] PBS or MPTP alone, MPTP and SPC from N-4YSyn immunized donors(MPTP+N-4YSyn), [bottom row, L to R] PBS and SPC from N-4YSyn immunizeddonors (PBS+N-4YSyn), MPTP and SPC from 4YSyn immunized donors(MPTP+4YSyn), and MPTP and T cells from N-4YSyn immunized donors(MPTP+N-4YSyn T Cells). Tissues collected 28 days post-MPTP treatment.FIG. 7B provides the counts of nigral Niss1+(left bars), TH+(centerbars), and TH− (right bars) neurons on day 28 after MPTP treatment asdetermined by stereological analysis. Control groups included micetreated with PBS alone (n=4), MPTP alone (n=8), and PBS animals thatreceived immune effector SPC from N-4YSyn immunized donor micePBS/N-4YSyn/SPC (n=6). Experimental groups included MPTP/PBS/SPC (n=6),MPTP/4YSyn/SPC (n=8), MPTP/N-4YSyn/SPC (n=9), and MPTP mice whichreceived purified T cells from N-4YSyn vaccinated donors. Values aremeans±SEM. P<0.01 compared to the following treatment groups: ^(a)PBS,^(b)MPTP, ^(c)MPTP/4YSyn/SPC.

FIG. 8A provides a scheme for immunization, lymphocyte proliferationassessment, and adoptive transfer of donor SPC in B6 mice. B6 (H-2b)mice were immunized with 10 μg 4YSyn in PBS, 50 μg N-4YSyn in CFA, 10 μgN-4YSyn in PBS or PBS in CFA. Mice were boosted 14 days later with theirrespective antigens as formulated previously with or without IFA. After5 days, single lymphoid cell suspensions were prepared and assessed forantigen-specific responses in standard lymphocyte proliferation assays.Single cell suspensions were pooled and adoptively transferred toMPTP-treated syngeneic recipients 12 hours after the final MPTPinjection. 5×10⁷ donor immune SPC were adoptively transferred toMPTP-treated recipient mice. Survival of dopaminergic neurons in the SNof recipient mice were evaluated after 7 days. FIG. 8B demonstratesantigen specific proliferation of SPC from B6 (H-2b) mice (n=5/group)immunized with PBS/CFA or N-4YSyn/CFA, and cultured for 5 days in mediaalone (left bars) or in the presence 1 μg/ml of 4YSyn (center bars) orN-4YSyn (right bars). Cultures were pulsed for 18 hours, cells harvestedand ³H-thymidine incorporation counted by β-scintillation spectrometry.Values represent mean stimulation indices6SEM and analyzed by ANOVA andBonferroni post-hoc tests. ^(a)p=0.0478.

FIGS. 9A and 9B demonstrate that lymphocytes from N-4YSyn immunizationexacerbate nigral dopaminergic neuronal loss in B6 mice. All panels ofFIG. 9A show TH+ neurons in the SN from mice treated with PBS or MPTPalone, MPTP and SPC from 4YSyn immunized donors (MPTP+4YSyn), MPTP andSPC from N-4YSyn immunized donors (MPTP+N-4YSyn) and lastly, MPTP andSPC from N-4YSyn+CFA immunized donors (MPTP+N-4YSyn CFA). FIG. 9Bprovides the counts of nigral TH+ and TH2 neurons on day 7 after MPTPtreatment. Experimental groups included mice treated with PBS alone(n=7), MPTP alone (n=7), MPTP/4YSyn (n=7), MPTP/N-4YSyn/CFA (n=6) andMPTP/N-4YSyn (n=6). Values are means±SEM. Analysis by ANOVA withBonferroni post-hoc tests indicated. ^(a)p<0.0001 compared to PBScontrol; ^(b)p<0.001 compared to MPTP group; ^(c)p<0.001 compared toMPTP/4YSyn group; and ^(d)p<0.03 compared to MPTP/4YSynCFA SPC.

FIG. 10 demonstrates N-4YSyn-mediated inhibition of T cellproliferation. Proliferative responses of anti-CD3 stimulated T cellsfrom naive B6 mice in presence of graded concentrations of 4YSyn orN-4YSyn (1, 3, 10, 30 μg/ml) or in media alone (0 μg/ml). T cells werecultured for 72 hours and pulsed with ³H-thymidine for the final 18hours of culture. Harvested cells were counted for ³H-thymidine uptakeby β-scintillation spectrometry and proliferation was expressed as meancounts per min (CPM)±SEM for quadruplicate samples and evaluated byANOVA with Bonferroni post-hoc tests. ^(a)p<0.01 compared with T cellsstimulated with anti-CD3 and cultured in media alone.

FIG. 11 demonstrates N-4YSyn activated immune SPC inducesmacrophage-mediated dopaminergic cell death. Representative fluorescencephotomicrographs are shown of live and dead cells from 24 hourmacrophage/MES 23.5 co-cultures in the presence of media alone, N-4YSyn,N-4YSyn and antigen-stimulated SPC of N-4YSyn-immunized mice(N-4YSyn+SPC), or N-4YSyn and the supernatants from antigen-stimulatedSPC of N-4YSyn-immunized mice (N-4YSyn+SPC Sup). Antigen-stimulated SPCfrom N-4YSyn immune mice were induced in vitro with N-4YSyn, and cellsand supernatants for use in the assay were harvested after 5 days ofculture. Controls included macrophages cultured in the presence of SPCsupernatants from antigen-stimulated SPC of N-4YSyn-immunized mice(Macrophages+SPC Sup); macrophages cultured in the presence of N-4YSynalone (Macrophages+N4YSyn); and Transwell™ cultures of plated MES 23.5cells and macrophages in the Transwell™ stimulated with N-4YSyn.Frequencies (±SEM) of dead cells for 4-8 fields/assay are provided inthe lower right corner of each panel. Differences in the meanfrequencies of dead cells were evaluated by ANOVA and Bonferronipost-hoc tests. p<0.01 compared to cultures treated with ^(a)Media,^(b)N-4YSyn, or ^(c)N-4YSyn+SPC.

FIGS. 12A-12C demonstrate CD4+ T cells modulate NF-κB activation inN-α-syn-stimulated microglia. Microglia were pretreated without or withCD4+ T cells, and NF-κB activity was assessed following 90 minutes ofstimulation with N-α-syn. FIG. 12A provides photomicrographs ofimmunofluorescent detection for NF-κB p65 in stimulated microglia (scalebar: 25 μm) and analysis for MFI per cell. Arrows indicate areas wherecolocalization of NF-κB p65 and nuclei appears to have occurred. FIG.12B provides Western blot analysis of nuclear fractions from stimulatedmicroglia with Abs to the NF-κB subunits p50/RELA (top panel), p65/NFκB1(middle panel), or a control Gapdh Ab (bottom panel). Mean OD wasnormalized to Gapdh expression. In addition, cDNA prepared from RNAisolated from duplicate samples was assessed by qPCR for expression ofNF-κB-related genes Tnfa, Tnfrs1a, Rela, and Nos2 (FIG. 12C) andneurotrophins Bdnf and Gdnf (FIG. 12D). Mean expression levels shownwere normalized to Gapdh expression. For FIGS. 12B and 12C, error barsrepresent SEM. Value of p<0.05 compared with media alone (CON; a),N-α-syn (b), or N-α-syn/Teff (c).

FIGS. 13A-13F demonstrate the inhibition of proinflammatorycytokine/chemokine production requires both cell contact and solublefactors. FIG. 13A shows cytokine/chemokine levels in microglial culturesupernatants treated with media alone, or N-α-syn without or withpretreatment or post-treatment with CD3-activated Treg or Teff weremeasured by cytometric bead array. Microglia were also assessed by flowcytometry for surface expression of CD206 (FIG. 13B) and MHC class II(FIG. 13C). Alternatively, FITC-conjugated latex beads were added to themicroglia cultures 30 minutes before flow cell analysis to evaluatephagocytosis by the MFI of microglia that phagocytized beads (FIG. 13D).Value of p<0.05 compared with microglia cultured with media alone (a),N-α-syn (b), or N-α-syn/Teff (c; FIGS. 13A-13D). Microglia were culturedwithout or with Treg either in direct contact or separated byTranswells™. Neutralizing Abs to IL-10, TGF-β, and CTLA-4 were added totandem direct contact cultures of microglia without and with Treg.Cytokine/chemokine concentrations (IFN-γ, TNF-α, IL-12, IL-6, MCP-1, andIL-10) in culture supernatants were determined by cytometric bead array(FIG. 13E) or ELISArray (IL-1α and IL-1β; FIG. 13F). Value of p<0.01compared with microglia cultured with N-α-syn (a) or with N-α-syn/Treg(b) in direct contact (FIGS. 13E and 13F). Error bars represent SEM.

FIGS. 14A-14D provide an analysis of microglial proteome. FIG. 14Aprovides fluorescence 2D DIGE and Decyder analysis of N-α-syn-stimulatedmicroglial cell lysates compared with unstimulated microglia. To assessphenotypic change following interaction with Treg, representative 2Dgels and Decyder analysis were performed on microglial cell lysatesassessing microglia cocultured with CD3-activated Treg beforestimulation with N-α-syn (FIG. 14B, pretreatment) or added in tandem 12hours following the addition of N-α-syn to the cultures (FIG. 14C,posttreatment). FIG. 14D provides Western blot analyses and volumetricand area intensity plot analyses by BVA for select proteins identifiedby LC-MS/MS are shown for cell lysates of unstimulated or N-α-synstimulated without or with pretreatment with Treg or Teff andposttreatment with Treg or Teff: L-plastin (spot: 23), ferritin L chain(spot: 28), peroxiredoxin 1 (spot: 65), and cathepsin D (spot: 13).

FIGS. 15A-15J demonstrate CD4+ T cells modulate microglial oxidativestress and CB activity. FIG. 15A provides confocal photomicrographs ofintracellular ROS production in microglia after 90 minutes ofstimulation with media (CON) or N-α-syn without and with T cellpretreatment (scale bar: 25 μm). FIG. 15B provides the MFI of ROSproduction per cell. FIG. 15C shows microglial intracellular GSHconcentration following 24 hour of exposure to N-α-syn without and withT cell pretreatment. Value of p<0.01 compared with microglia culturedwith media alone (CON; a), N-α-syn (b), or N-α-syn/Teff (c; FIGS. 15Band 15C). Western blot analyses of select redoxactive proteinsidentified by LC-MS/MS, including THX 1 (spot: 49; FIG. 15D), BVR B(spot: 34; FIG. 15E), HSP 70 (spot: 3; FIG. 15F), and GLU 1 (spot: 64;FIG. 15G). CB activity in microglia after stimulation with N-α-syn for24 hour is demonstrated by fluorescence photomicrographs (scale bar: 25μm; FIG. 15H) and MFI analysis (FIG. 15I). Value of p<0.05 compared withmicroglia cultured to media alone (CON; a), N-α-syn (b), or N-α-syn/Teff(c). FIG. 15J provides representative Western blot analysis ofmicroglial lysates and culture supernatants for CB [spot: 27] expressionand reprobed with Ab against β-actin following pretreatment with Treg orTeff and stimulation for 24 hours with N-α-syn. Error bars representSEM.

FIGS. 16A-16I demonstrate Treg induce microglial apoptosis throughFas-FasL interactions. FIG. 16A provides Western blot analysis forcaspase-3 (procaspase-3 and cleaved) expression in microglial celllysates from unstimulated (lane 1) and N-α-syn stimulated alone (lane2), pretreated with Treg or Teff (lanes 3 and 4), or post-treated withTreg or Teff (lanes 5 and 6). FIG. 16B provides flow cell analysis forMFI of active caspase-3 expression by microglia. Value of p<0.05compared with media alone (a) and N-α-syn stimulation (b). FIG. 16Cprovides confocal photomicrographs and MFI for active caspase-3 on a percell basis (scale bar: 25 μm). Value of p<0.01 compared with microgliacultured in media alone (CON; a), N-α-syn stimulation alone (b), orN-α-syn/Teff (c). FIG. 16D provides flow cell analysis for FasLexpression by Treg or Teff immediately following isolation (naive Tcells), following CD3 activation (anti-CD3 T cells), and after coculturewith N-α-syn-stimulated microglia for 24 hours (post-coculture). Meanpercentages of FasL+CD4+ T cells shown [value of p<0.05 compared withnaive T cells (a) and αCD3 T cells (b)]. FIG. 16E provides flow cellanalysis of Fas expression by microglia treated for 24 hours without andwith N-α-syn stimulation and T cell post-treatment. Percentages of Fas+cells are shown [value of p<0.05 compared with media alone (a) andN-α-syn stimulation alone (b)]. FIG. 16F provides an MTT assay to assessmicroglial susceptibility to spontaneous- and anti-CD95-inducedapoptosis after culture for 24 hours in media alone and N-α-syn withoutor with T cell pretreatment. Value of p<0.05 compared with media alone(a), N-α-syn stimulation alone (b), N-α-syn/Teff (c), media alone withanti-CD95 stimulation (d), and N-α-syn with anti-CD95 stimulation (e).FIG. 16G provides a TUNEL assay of microglia treated with media (CON)and N-α-syn without and with post-treatment with Treg or Teff in theabsence or presence of anti-FasL. Photomicrographs (scale bar: 25 μm)and MFI of TUNEL+ cells normalized to the number of DAPI-stained nuclei.FasL dependence in Treg-induced apoptosis of stimulated microglia wasalso assessed by MTT assay (H) and caspase 3/7 activity assays (FIG.16I). Values shown as a percentage of unstimulated controls (MTT) or MFI(caspase-3/7 activity). Value of p<0.05 compared with media alone (a),N-α-syn stimulation alone (b), N-α-syn/Teff (c), and post-treatmentwithout anti-FasL (d; FIGS. 16G-16I). Error bars represent SEM.

FIG. 17A-17D demonstrate CB regulates microglial apoptosis. FIG. 17Aprovide Western blot analysis for CB and Gapdh expression in microglialcell lysates following treatment with media (CON) or N-α-syn without andwith Treg or Teff after N-α-syn stimulation (post-treatment). FIG. 17Bprovides confocal photomicrographs (scale bar: 25 μm) and MFI per cellof active caspase-3 expression in N-α-syn-stimulated microglia in thepresence or absence of Treg or Teff in the absence or presence of acell-permeable CB inhibitor (CA-074 Me). The MTT assay (FIG. 17C) andcaspase 3/7 activity assay (FIG. 17D) of microglia also revealed thatinhibition of CB significantly diminished stimulation-induced apoptosis.Values shown are means (±SEM) of absorbance as a percentage ofunstimulated controls (MTT) or MFI (caspase-3/7 activity). Value ofp<0.05 compared with media alone (a), N-α-syn stimulation alone (b),N-α-syn/Teff (c), post-treatment without CA-074Me (d), and N-α-syn withCA-074Me (e).

FIG. 18 provides the experimental design for microglial proteomicsprotein discovery. Microglia were co-cultured for 24 hours with CD4+CD25+Treg (or Teff) or without as control. Treg (or Teff) were removedfrom the cultures and the microglia stimulated with aggregated N-α-synfor 24 hours [pre-treatment] to represent asymptomatic disease (A).Alternatively, microglia were stimulated with N-α-syn for 12 hours priorto the addition of Treg [post-treatment] to represent more overt disease(B). Twenty-four hours later microglial cell protein lysates wereprepared and subjected to 2D electrophoresis. Decyder™ analysis softwarewas used to match spots and identify expression patterns. Selectedprotein spots were excised, digested with trypsin and identified bynano-LC-MS/MS peptide sequencing. Database searches were performed usingSEQUEST with criteria thresholds set to afford greater than 95%confidence level in peptide identification.

FIG. 19 provides 2D-DIGE of proteins from all experimental groups withmatched spots picked for sequencing identifications by nano-LC-MS/MS. Arepresentative preparative gel is shown. Equal amounts of protein werepooled from all experimental groups (unstimulated, N-α-syn-stimulated,pre- and posttreatment with Treg or Teff) and replicates for a totalconcentration of 450 μg. The pooled sample was applied to a pH gradientstrip and separated with isoelectric focusing for the first dimension.For the second dimension, the strip was loaded onto a large formatgradient gel and separated based on molecular weight. Followingelectrophoresis, the gel was fixed and post-stained with Deep Purple forpositive detection of protein spots. Circled spots identified by BVAusing Decyder™ analysis software were selected for excision. Proteinswith the most peptides positively identified within a specific spot arelabeled on the gel. (Abbreviations: Prdx, peroxiredoxin; Thx,thioredoxin, Vdac, voltage-dependent anion channel; Sod, superoxidedismutase; Hsp, heat shock protein; Capg, macrophage capping protein;NAD, nicotinamide adenine dinucleotide).

FIGS. 20A-20F provide the classification of proteins modulated byN-α-syn stimulation and Treg treatment. Pie-chart diagrams represent theproportion (%) of proteins within specific categories based onclassification and function identified by mass spectrometry. FIG. 20Aprovides classification of proteins differentially expressed bymicroglia in response to N-α-syn stimulation alone (clockwise from 17%segment: redox-active, proteases, chaperones, UPS, glycolysis,transcription, cell motility, structural, apoptosis, metabolism, ox.phospho.). FIG. 20B provides relative expression of proteins in responseto N-α-syn stimulation compared to unstimulated controls. Severalproteins within each category showing both increased and decreasedproteins were identified including those for apoptosis (*gelsolinincreased; nucleoside-diphosphate kinase decreased) and glycolysis(^(§)enolase 3 and lactate dehydrogenase increased; alpha enolase,pyruvated dehydrogenase, pyruvate kinase, and triosphosphate isomerase 1decreased). FIG. 20C provides proportion of microglial proteinsdifferentially expressed in response to N-α-syn following Tregpre-treatment and the relative expression trends shown in FIG. 20D.Categories associated with transcription (*VIP-receptor gene repressorprotein, TAR DNA binding protein, and Ubiquitin conjugating enzyme E2Nincreased; MRG-binding protein decreased), cell motility(^(§)microtubule associated protein increased; laminin B2, beta actin,and alpha tubulin decreased), structural (^(#)Capg and guaninenucleotide exchange factor increased; vimentin, cofilin 1 and 2decreased), and oxidative phosphorylation (^(†)NADH dehydrogenase Fe—S,ATP synthase O subunit, H+-ATP synthase e subunit, and cytochrome coxidase increased; ATP synthase F0 complex decreased) consisted of bothincreased and decreased expression of proteins. FIG. 20 E provides theproportion of microglial proteins differentially expressed in responseto N-α-syn following Treg post-treatment and the relative expressiontrends are shown in FIG. 20F. Categories associated with metabolism(^(#)phosphoglycerate mutase 1 increased; aldolase 1 and aldehydedehydrogenase 2 decreased), oxidative phosphorylation (§ATP synthase Dincreased; H+-transporting two-sector ATPase alpha chain decreased), andchaperones (^(#)cyclophilin A increased; protein disulfide isomerasedecreased) consisted of both increased and decreased expression ofproteins.

FIGS. 21A-21D demonstrate that Treg modulate microglial inflammation toattenuate the neurotoxic phenotype of N-α-syn stimulated microglia. FIG.21A provides photomicrographs (20× magnification) of Prx1 expression inmicroglia treated with media alone (CON), N-α-syn, or cultured with CD4+T cell subsets following pre- and post-treatment. Values shown are themean fluorescence intensity (MFI) per field±SEM. FIG. 21B providesWestern blot analysis for α-tubulin, galectin 3 and gelsolin in responseto treatment normalized to Gapdh expression within the same blot forcomparisons. FIG. 21C provides photomicrographs (20× magnification) ofactin expression or Hsp70 in microglia treated with media alone (CON),N-α-syn, or cultured with CD4+ T cell subsets following pre- andpost-treatment. Values shown are the MFI per field±SEM. FIG. 21Ddemonstrates the survival of MES23.5 cells after co-culture with N-α-synstimulated microglia with and without Treg or Teff or after culture withcondition media (supernatants) of N-α-syn stimulated microglia treatedwith either Treg or Teff. Values SEM (P<0.01 vs. ^(a)CON, ^(b)N-α-synalone, ^(c)N-α-syn/Teff).

FIGS. 22A-22G provide the N-α-syn stimulated proteome. FIGS. 22H-22Mprovide the modulation of the N-α-syn microglial proteome by Tregpretreatment. FIGS. 22N-22Q provide the modulation of the N-α-synmicroglial proteome by Treg post-treatment *The CID spectra werecompared against those of the EMBL nonredundant protein database byusing SEQUEST (ThermoElectron, San Jose, Calif.). After filtering theresults based on cross correlation Xcorr (cutoffs of 2.0 for [M+H]1+,2.5 for [M+2H]2+, and 3.0 for [M+3H]3+), peptides with scores greaterthan 3000 and meeting delta cross-correlation scores (·Cn)>0.3, andfragment ion numbers >60% were deemed valid by these SEQUEST criteriathresholds, which have been determined to afford greater than 95%confidence level in peptide identification. ⁵⁵⁴ SwissProt accessionnumber (accessible at ca.expasy.org/sprot/). ^(‡)International ProteinIndex (IPI) (accessible at www.ebi.ac.uk/IPI/). §Theoretical molecularmass for the primary translation product calculated from protein DNAsequences. ^(II)Theoretical isoelectric point. ^(¶)Postulatedsubcellular location (accessible at locate.imb.ug.edu.au).^(#)Postulated cellular function (accessible at ca.expasy.org/sprot/).**Number of different peptides identified for each protein. ^(††)(FIGS.22A-22G) Fold changes of proteins in N-α-syn stimulated microgliallysates versus unstimulated microglial lysates. Negative DIGE indexindicates decreased expression in N-α-syn stimulated microglia relativeto controls. ^(††) (FIGS. 22H-22M) Fold changes of proteins in Tregpre-treated microglia versus N-α-syn alone stimulated microgliallysates. ^(††) (FIGS. 22N-22Q) Fold changes of proteins inTreg-post-treated microglia versus N-α-syn alone stimulated microgliallysates. ^(‡)P-values as determined by Biological Variation Analysis byone-way ANOVA for pair-wise comparison between treatments.

FIGS. 23A-23E demonstrate α-Syn nitration, aggregation, and microglialactivation. FIG. 23A is a Coomassie stain of anti-N-α/β-synucleinimmunoprecipitation from SN from control and PD brains. Arrowheadreflects the area excised from gel and submitted for LC-MS/MS analysis.Equal concentrations of proteins from control and experimental braintissues served as loading controls. Peptides obtained by LC-MS/MS thatmatched human α-syn are highlighted within the full-length sequence (SEQID NO: 13). FIG. 23B is a western blot analyses of recombinant mouseα-syn and derivatives. Lane 1 is a nitrotyrosine modified proteinprovided by the manufacturer. Lanes 1-3 were blotted and probed withanti-nitrotyrosine, and lanes 4-6 were probed with anti-synuclein. FIG.23C are AFM images are shown for unaggregated (0.4×0.4 mm) andaggregated N-α-syn (1.6×1.6 mm). Arrow indicates location of insetphotomicrograph. Scale bar corresponds to height of aggregates on theinterface. FIG. 23D shows microglial morphology after exposure ofmicroglia to media alone (control, left) or 100 nmol/L N-α-syn (center),and N-α-syn stimulated microglia in co-culture with MES23.5 cells(right; scale bar: 25 μm). Cells were stained with calcein AM to detectviable cells. FIG. 23 E provides cytokine bead arrays were used for flowcytometric analysis of supernatants from unstimulated microglia(control, open box) and microglia stimulated with either 100 nmolILN-α-syn (closed triangle) or 100 ng LPS (closed circle) (n=3, p<0.01 VS.^(a)control and ^(b)LPS at each corresponding time point).

FIGS. 24A-24E demonstrate N-α-syn-stimulated microglia decreasedopaminergic cell survival. FIG. 24A is representative photomicrographsof Live/Dead assays of unstimulated or N-a-syn stimulated microgliaco-cultured with MES23.5 cells for 24 hours (scale bars: 25 μm). FIGS.24B and C are graphs of N-α-syn-induced microglial inhibition of cellsurvival. A time-course for cell survival is shown for MES23.5 cells andmicroglia co-cultured in the presence of media alone (Con, box), 100nmol/L unmodified a-synuclein (α-syn, triangle), or 100 nmol/LN-α-synuclein (N-α-syn, circle). Cell viability was quantified using theLive/Dead assay by (FIG. 24B) cell count (n=9 fields, p<0.01 comparedwith a 0 hour and ball treatment groups at corresponding time point),and by (FIG. 24C) fluorometric analysis (n=9 fields, p<0.01 comparedwith ^(a)0 hour and ^(b)all treatment groups at corresponding timepoint). FIG. 24D provides cell survival of MES23.5 cells in co-culturewith microglia after 72 hours of stimulation with either α-syn orN-α-syn (n=9, p<0.01 compared with ^(a)all treatment groups and^(b)α-syn stimulated microglia). FIG. 24E shows the influence ofsecretory factors from microglia stimulated with either α-syn or N-α-synfor 24 hours on MES23.5 cells was determined. Cell survival was assessedfollowing incubation with supernatants or in Transwell™ format for 24hours (n=3, p<0.01 compared with ^(a)all treatment groups and^(b)α-syn-stimulated microglia).

FIG. 25 demonstrates cellular activation and oxidative stress pathwaysin PD brain tissues. Tissue samples from the SN and BG of control(filled bars) and PD patients (open bars) were evaluated by qRT-PCR forexpression of NF-κB pathway associated genes. The relative expression ofa gene was normalized to GAPDH in the same sample and values arerepresented as mean±SEM (^(a)p<0.05, ^(b)p<0.01, and ^(c)p<0.001compared with samples from control patients, n=8-10 patients per group).

FIG. 26 shows NF-κB translocation in PD. Expression of NF-κB subunitsp50/NFKB1 and p65/RELA proteins were evaluated by western blot analysisfrom whole tissue lysates (top), cytosolic fractions (middle), andnuclear fractions (bottom) of SN from control and PD patients.Expression of phosphorylated RELA/p65 [NF-κB pS536] within the nuclearfraction was also assessed. The mean densitometric values weredetermined with IMAGEJ software and normalized to GAPDH expression inthe same sample (bottom). Values are represented as the mean density±SEMfor four patients/group and p-values of Student's t-test for pair-wisecomparisons of densities from control (open bars) and PD (filled bars)patients are *p<0.05 and **p<0.005. Blots are representative of twoindependent experiments (n=4 patients per group).

FIG. 27 provides microarray analysis of N-α-syn-stimulated microglia.RNA was isolated from microglial cells stimulated with 100 nmol/LN-α-syn or 100 ng/mL LPS from which cDNA was made and amplified. FIG.27A provides general pathway-focused microarray revealed involvement ofNF-κB signaling pathways. FIG. 27B provides focused arrays were utilizedfor regulation of NF-κB associated genes for microglia that wereunstimulated (0 hour Control) or stimulated with 100 nmol/L N-a-syn or100 ng/mL LPS for 1 hour and 4 hours, respectively. Boxes indicate genesthat were induced by stimulation at 1 and 4 hours. FIG. 27C providesgraphs of the qPCR of mRNA from samples confirmed representativeinductions for genes (rank and file position in microarray) Ccl2 (F2),I11b (H5), Tnfrsf1a (D13), Stat1 (11E), Rela (10F), Tnf (A13), and Nos2.Gene expression for the neurotrophins Bdnf and Gdnf were also assessedby qPCR from the same mRNA/cDNA samples [n=3, p<0.01 compared with ^(a)0hour control (C) and ^(b)LPS at corresponding time point]. FIGS. 27D and27E provide N-α-syn- and LPS-stimulated microglial transcriptome. Valuesrepresent fold-change versus unstimulated controls. ^(b)NCBI EntrezGeneID. FIGS. 27F-27H provide N-α-syn-stimulated microglial proteome.^(a)The CID spectra were compared against those of the EMBLnon-redundant protein database by using SEQUEST (ThermoElectron, SanJose, Calif.). After filtering the results based on cross-correlationXcorr (cutoffs of 2.0 for [M+H] 1+, 2.5 for [M+2H]2+, and 3.0 for[M+3H]3+), peptides with scores greater than 3000 and meeting deltacross-correlation scores (DCn)>0.3, and fragment ion numbers >60% weredeemed valid by these SEQUEST criteria thresholds, which have beendetermined to afford greater than 95% confidence level in peptideidentification; ^(b)Theoretical molecular mass; ^(c)Isoelectric point;^(d)Accession numbers for UniProt (accessible atwww.ipr.uniprot.org/search/textSearch.shtml); ^(e)Hours followingstimulation with N-α-syn; ^(f)Number of peptides identified for eachprotein selected based on the above mentioned criteria; ^(g)Volume ratioindicates fold-change versus control.

FIGS. 28A-28C provide 2DE and LC-MS/MS analysis of theN-α-syn-stimulated microglia proteome. Fluorescence 2D DIGE analysis ofN-α-syn-activated microglial cell lysates. Fluorescence 2D DIGE (2DE)analysis of activated microglial cell lysates at 2, 4, and 8 hours afterN-α-syn stimulation. Proteins from cell lysates of unstimulatedmicroglia labeled with Cy3 appear green on the 2 dimensional gels, whileproteins of N-α-syn stimulated microglia labeled with Cy5 appear red,and proteins common to both appear yellow. Three-dimensional DeCyderinterpretation for six representative proteins per time-point are shown.The numbers correspond to the protein spot labeled on gels. Analysis ofspot distribution to locate and define protein spots (right panel).Protein spots from samples of stimulated cell lysates were identified asdecreased, increased, or common versus non-stimulated cell lysates.Spots picked for sequencing analysis with LC-MS/MS are shown.Abbreviations: HSP70, heat-shock protein 70; Cyt c oxidase, cytochrome coxidase; SOD, superoxide dismutase. A complete listing of all proteinsidentified through 2DE is contained within FIG. 27.

FIG. 29 provides N-α-syn-stimulated microglial proteins in PD braintissue. Immunoblot identification of proteins in the SN and BG of PDbrains that were previously observed in N-α-syn-stimulated microglia.This includes 14-3-3σ, calmodulin, galectin-3, L-plastin, actin,tubulin, glutathione-S-transferase, thioredoxin, and biliverdinreductase. The proteins are divided into regulatory, cytoskeleton, orredox functions. The mean densitometric values were determined withIMAGEJ software and normalized to GAPDH expression in the same sample(bottom). Values are represented as the mean density±SEM for fourpatients/group and p-values of Student's t-test of pair-wise comparisonsof densities from Control (open bars) and PD (closed bars) patients are*p<0.05 (**p<0.05 and congruent results with the N-a-syn-microglialproteomic and western blot assays).

FIGS. 30A-30C provide SELDI-TOF, 1D SDS PAGE, and Western blot analysesof supernatant fluids obtained from N-α-syn-activated microglial.Representative SELDI-TOF spectral analysis (region 10-20 kDa) ofuntreated-control (top panel) and N-α-syn-stimulated microglia (bottompanel) at 16 hours post-stimulation, shown in FIG. 30A. Marked by anasterisk are upregulated and uniquely expressed peaks corresponding inmolecular weight within 1% of mass tolerance to proteins identified byLC-MS/MS. These include calcyclin (10,051 Da), thioredoxin (11,544 Da),calvasculin (11,721 Da), calmodulin (16,706 Da), and TNF-α (17,907 Da).Bands were excised from 1D SDS PAGE gel, digested by trypsin, andsequenced by LC-MS/MS. Lanes are supernatant fluids obtained fromcontrol (unstimulated) microglial=[Lanes 1-3] and supernatants fromN-α-syn-activated microglia [Lanes 4-6] collected 8 hours, 16 hours, and24 hours post-stimulation, respectively (FIG. 30B). RepresentativeWestern blots of supernatant fluids from control and N-α-syn-stimulatedmicroglia 16 hours post-stimulation for proteins identified by LC-MS/MS(FIG. 30C). FIGS. 30D and 30E provide the secretome ofN-α-syn-stimulated microglia. **The CID spectra were compared againstthose of the EMBL nonredundant protein database by using SEQUEST(ThermoElectron, San Jose, Calif.). After filtering the results based oncross correlation Xcorr (cutoffs of 2.0 for [M+H] 1+, 2.5 for [M+2H]2+,and 3.0 for [M+3H]3+), peptides with scores greater than 3000 andmeeting delta cross-correlation scores (Cn)>0.3, and fragment ionnumbers >60% were deemed valid by these SEQUEST criteria thresholds,which have been determined to afford greater than 95% confidence levelin peptide identification. ^(a)Theoretical molecular mass.^(b)Isoelectric point. ^(c)Accession numbers for UniProt (accessible atwww.ipr.uniprot.org/search/textSearch.shtml). ^(d)Number of peptidesidentified for each protein selected based on the above mentionedcriteria. Proteins were considered if 2 or more peptides wereidentified. ^(e)Proteins were increased or decreased in supernatants ofmicroglia stimulated with N-α-syn for 4 hours when compared tounstimulated microglia (controls).

FIG. 31 provides N-α-Syn activated microglial metabolic responses.Microglia were stimulated with N-α-syn over the course of 24 hours andculture supernatants were collected at specified times followingstimulation for analysis of extracellular metabolites, shown in FIG.31A. Analysis of intracellular metabolites following engagement withN-α-syn is shown in FIG. 31B. Mean metabolite concentrations arepresented as values±SEM, and are representative of three separateexperiments, (*P<0.001, v. unstimulated control).

FIG. 32 shows cathepsin B expression and functional activity followingN-α-syn-microglial activation. Comparative analysis of cystatin B andcathepsin B protein expression in microglia at several time pointsduring stimulation with aggregated N-α-syn by western blot, shown inFIG. 32A. Mean density of protein bands (±SEM) were normalized to GAPDHexpression on the same blot (*P<0.05 compared to unstimulated microgliaat 0 hour) (FIG. 32B). Cathepsin B enzymatic activity prior tostimulation (0 hour) and at 2 hours, 4 hours, 8 hours, and 24 hoursafter stimulation with N-α-syn. Enzymatic activity is visualized withthe red fluorogenic substrate, CV-(RR)₂ and nuclei are stained blue withHoechst dye. Fluorescence intensities were determined by ImageQuant andare presented as mean±SEM for n=3 fields for 4 replicates (P<0.05compared to mean at 0 hour).

FIG. 33 shows the effect of Cathepsin B inhibition on N-α-syn-mediatedcytotoxicity. Supernatants from N-α-syn stimulated microglia inducedsignificant DA cell death. Whereas, inhibition of cathepsin B activityby either the cell impermeable inhibitor CA-074 or the cell-permeableinhibitor CA-074 Me resulted in partial protection from N-α-syn mediatedDA cell death, shown. Values are shown as mean dead DA cells±SEM for n=6replicates per treatment paradigm.

FIG. 34 shows nuclear translocation of NF-κB subunits. Cytosol (top) andnuclear (bottom) fractions were prepared from microglia stimulated withN-α-syn for subsequent time-points and assessed for expression of NF-κBsubunits NFκB1/p50 and RelA/p65 by western blot (FIG. 34A). Mean densityof protein bands for NFκB1/p50 (FIG. 34B) and RelA/p65 (FIG. 34C) bywestern blot were normalized to GAPDH in the same sample. Values areshown mean±SEM for n=3 replicates per time point (*P<0.01 compared to 0hour).

FIG. 35 shows N-α-syn adaptive immunity accelerates MPTP-nigrostriataldegeneration. FIGS. 35A-35C provide photomicrographs (scale bar 25 μm)and enumeration of Mac-1+, FJ-C+, and TH+ cells, respectively, in theSNpc of mice treated with PBS, MPTP, or MPTP and N-4YSyn SPCs. FIG. 35Ashows Mac-1+ reactive microglia per mm². FIG. 35B provides total numbersof FJ-C+ neurons. FIG. 35C shows dopaminergic neurons in the SNpcidentified as TH+Niss1+ neurons (black bars), while non-dopaminergicneurons were identified as TH-Niss1+ neurons (gray bars). Differences inmeans (±SEM) were determined where P<0.05 compared with groups treatedwith ^(a)PBS or ^(b)MPTP. FIG. 35D provides images demonstrating thatMPTP-intoxicated recipients of N-4YSyn SPC had increased infiltration ofCD4+ cells within the SNpc following adoptive transfers, whereasMPTP-intoxication alone showed limited infiltrates at 48 hours-postintoxication and no CD4+ cells were identified in PBS-treated controls.FIG. 35E is a graph demonstrating that T cells isolated from N-4YSyndonors stimulated in vitro with anti-CD3 for 24 hours produced greaterconcentrations of IL-17a and TNF-α relative to naïve T cells. FIG. 35Fis a graph demonstrating that N-4YSyn antigenic stimulation of CD4+effector T cells isolated from immunized mice induced the production ofcertain cytokines. FIG. 35G is a graph of the capacity of Treg isolatedfrom immunized FoxP3-GFP mice to inhibit effector T cell proliferationto anti-CD3 stimulation following immunization with N-4YSyn (20%) ascompared to Treg isolated from naïve donors (80%) at a ratio of 1:1.

FIG. 36A is a schematic of the adoptive transfer protocol. FIG. 36Bprovides graphs demonstrating a cytometric bead array analysisconfirming that the CD4+ T cell subtypes were indeed polarized to thedesignated phenotype. FIG. 36C provides images of TH immunostainedventral midbrain and striatum 7 days after MPTP treatment and adoptivetransfers. The adoptive transfer of both N-4YSyn Th1 and Th17 subsetsresulted in decreased numbers of surviving TH+ neurons within the SNpc;whereas, only N-4YSyn Th17 cells resulted in diminished TH terminidensity within the striatum. FIG. 36D is a graph showing that theadoptive transfer of N-4YSyn Th17 cells induced a 53% decrease in thenumber of surviving TH+ neurons relative to MPTP-intoxication alone.FIG. 36E is a graph of the TH density within the striatum. Adoptivetransfer of N-4YSyn Th17 cells significantly exacerbated theMPTP-induced loss of striatal TH density to 5% of PBS-treated controls.

FIG. 37 shows microglial activation and dopaminergic neuroprotection anddegeneration. FIG. 37A provides photomicrographs (scale bar 25 μm) ofmidbrain immunostained for Mac-1 (top panel) or FJ-C to identify dead ordying neurons (bottom panel). FIG. 37B provides the mean numbers ofMac-1+ microglia and FIG. 37C provides FJ-C+ neurons within the SNpc.Differences in means (±SEM) were determined where P<0.05 compared togroups treated with ^(a)PBS, ^(b)MPTP, ^(c)MPTP+N-4YSyn SPC, or^(d)MPTP+VIP SPC.

FIG. 38 shows Treg mediated dopaminergic neuroprotection inN-α-syn-immunized MPTP-intoxicated animals. FIG. 38A shows midbrainsections (scale bar 25 μm) (top panel) and striatum (bottom panel)immunostained for TH. Dopaminergic neurons in the SNpc were identifiedas TH+Niss1+ neurons (left bars), while non-dopaminergic neurons wereidentified as TH-Niss1+ neurons (right bars) (FIG. 38B). Mean densitiesof striatal dopaminergic termini were determined by digital imageanalysis (FIG. 38C). Differences in means (±SEM) were determined whereP<0.05 compared to groups treated with ^(a)PBS, ^(b)MPTP, ^(c)MPTP+VIPSPC, ^(d)MPTP+N-4YSyn SPC. Dopaminergic neuronal survival followingadoptive transfer of 5×10⁷ N-4YSyn SPC with 1×10⁶ Treg (FIG. 38D). FIG.38E shows density of striatal dopaminergic termini. Differences in means(±SEM) were determined where P<0.05 compared to groups treated with^(a)PBS, ^(b)MPTP, ^(c)MPTP+N-4YSyn SPC, ^(d)MPTP+N-4YSyn+naive SPC, or^(e)MPTP+N-4YSyn+naïve Treg.

FIG. 39 shows phenotypic and functional characterization ofneuroprotective Treg. FIG. 39A shows immunization with N-4YSyn ortreatment with VIP altered the frequencies of splenic CD3+, CD19+, CD4+,and CD4+ CD25+ cells. FIG. 39B is a graph showing the ability of CD4+CD25+CD62L^(low) Treg isolated from naïve, N-4YSyn-immunized, andVIP-treated mice to inhibit CD3-mediated proliferation of CD4+CD25-naïve T cells. FIG. 39C is a table of relative fold-differences inexpression of CD4+ T cell related genes from T cells isolated fromN-4YSyn immunized, VIP-treated, and pooled T cells compared with T cellsfrom naïve mice. FIG. 39D is a graph of the cytokine production assayedfrom T cell supernatants, normalized to absorbance obtained fromsupernatants of naïve T cells. FIG. 39E is a graph of T cellproliferation to no antigen [media], 4YSyn, or N-4YSyn. FIG. 39F is adose response of N-4YSyn on T cell proliferation. FIG. 39G is graph ofthe fold difference to gene expression of N-4YSyn T cells.

FIG. 40 shows HIV-1/VSV-infected BMM induce HIVE pathology. Inflammatorypathological effects of virally infected BMM in brains of immunecompetent mice were evaluated. BMM infected with HIV-1NVSV weretereotactically injected via the i.c. route into the basal ganglia ofsyngeneic C57BL/6J mice. Brain tissues were dissected on day 7 and HIVEpathology was analyzed after immunostaining for expression of HIV-1 p24,anti-CD3, GFAP, and Iba1 Ags. Representative micrographs are shown atthe original magnification of ×100 for HIV-1 p24, CD3, GFAP, and Iba1and at ×400 for bone marrow cell and HIV-1 p24-positive BMM. Flowcytometric analysis demonstrated >95% CD11b+BMM.

FIG. 41 shows Treg attenuate chronic neuroinflammatory responses in HIVEmice. FIG. 41A shows a flow cytometric analysis of Treg and Teff subsetsfrom naïve C57BL/6 mice showing percentage distribution of the followingCD4+ T cell phenotypes: CD4+ CD25+, CD4+ CD25−, CD4+ FoxP3+, or CD4+FoxP3−. FIG. 41B provides a quantitative PCR analysis of mRNA encodingFoxP3, TGF-β, IL-10, IL-2, and IFN-γ from CD3-activated Treg (top bars)and Teff (bottom bars). Mean±SEM of mRNA levels was determined fortriplicate cell samples and normalized to GAPDH. Significant differencesin relative expression of mRNA from Treg compared with Teff weredetermined by Student's t test, *, p<0.05. FIG. 41C shows Treginhibition of anti-CD3-mediated proliferation of 1×10⁴ Teff. Cells werecocultured for 72 hours and pulsed with [³H]thymidine for the final 18hours of culture, harvested onto filters, and counted by betascintillation spectrometry. FIG. 41D are images of HIVE,HIV-1/VSV-infected BMM which were stereotactically injected i.c. intothe basal ganglia of syngeneic C57BL/6J mice. Sham controls wereinjected i.c. with PBS. Treg or Teff (1×10⁶) were adoptively transferredinto HIVE mice 1 day postinfection. Serial sections of brain tissue thatcomprise the injection area were obtained on day 7 postinfection andanalyzed by immunohistochemical and Western blot assays for p24, Iba1,Mac-1, GFAP, CD4, and FoxP3 Ags. Brains were collected at day 7 afteri.v. injections. Representative brain sections from PBS and theHIV-1/VSV, HIV-1/VSV/Teff, and HIV-1/VSV/Treg groups showing expressionof HIV-1 p24, Iba1, Mac-1, GFAP, CD4, and FoxP3. Where indicated, nucleiwere stained with DAPI (scale bars, 50 μm; original magnification,×400). Cellular colocalizations of intracellular HIV-1 p24 and membraneMac-1 expression are shown in magnified inserts. FIG. 41E providesdigital image quantification of fluorescence intensity in the stainedarea was analyzed under ×400 magnification using NIH Image J software.Eight fields in four sections in each experiment were subjected toquantitative analysis. Bar graphs represent mean of area stainedpositive intensities in a field of view (open bars, PBS; gray bars,HIV-1/VSV; speckled bars, HIV-1/VSV/Teff; or black bars,HIV-1/VSV/Treg). FIG. 41F shows representative Western blot analysis ofIba1, GFAP, TNF-α and β-actin levels from brains of mice treated withPBS, HIV-1/VSV, HIV-1/VSV/Teff (Teff), or HIV-1/VSV/Treg (Treg). FIG.41G shows densitometric quantification of Western blots for Iba1, GFAP,and TNF-α levels in mice treated with PBS (open bars), HIV-1/VSV (graybars), HIV-1/VSV/Teff (speckled bars), or HIV-1/VSV/Treg (black bars).Levels were normalized to β-actin levels and mean densities±SEM weredetermined from four mice per group. FIGS. 41E and 41G, compared withPBS: *, p<0.05; **, p<0.01; p<0.001; and compared with HIV/VSV group: #,p<0.05; and ###, p<0.001.

FIG. 42 shows Treg are neuroprotective in HIVE mice. Mice werestereotactically injected into the basal ganglia with HIV-1/VSV-infectedsyngeneic BMM or with PBS alone as sham controls. After 1 day, Treg orTeff (1×10⁶) were adoptively transferred into HIVE mice. Serial sectionsof brain tissue that encompassed the injection area were obtained on day7 postinfection and analyzed by immunohistochemistry. FIG. 42A showsserial sections of brains from PBS control, HIV-1/VSV, HIV-1/VSV/Teff,and HIV-1/VSV/Treg were stained for MAP2, NeuN, BDNF and GDNF andvisualized by confocal laser-scanning microscopy. Images are shown atx400 original magnification and the scale bars equal 50 μm. FIG. 42Bshows stained sections from mice (eight fields in four sections in eachmouse) treated with PBS (open bars), HIV-1/VSV (gray bars),HIV-1/VSV/Teff (speckled bars), or HIV-1/VSV/Treg (black bars) whichwere digitally analyzed using NIH Image J software. Bar graphs showedmean of fluorescence intensity per field of view. FIGS. 42C and 42D showWestern blot analysis of BDNF expression (FIG. 42C) and densitometricquantification of BDNF Western blots (FIG. 42D) from lysates of braintissues from mice treated with PBS, HIV-1/VSV, HIV-1/VSV/Teff, orHIV-1/VSV/Treg. BDNF levels were normalized to β-actin expression.Values are expressed as mean±SEM for four mice per group and weresignificant compared with the PBS group: *, p<0.001; **, p<0.01; ***,p<0.001, and compared with HIV/VSV group: ##, p<0.01; and ###, p<0.001.

FIG. 43 shows Treg induce apoptosis in HIV-1/VSV-infected BMM.HIV-1/VSV-infected BMM were exposed to Teff or Treg for 3 days in theabsence of M-CSF at a ratio of 3:1 (BMM:Treg or BMM:Teff). UninfectedBMM and HIV-1/VSV-infected BMM served as negative and infectioncontrols. In FIG. 43A, after 3 days, cell viabilities were determined byMTT assay. For FIG. 43B, cell survival/cytotoxicity was assayed byLIVE/DEAD cytotoxicity immunostaining (Invitrogen) and visualized byfluorescent microscopy at ×400 original magnification. Scale bar equals50 μm. Mean percentages of cells±SEM were determined for threecultures/group and significant differences in means were determined byStudent's t test compared with uninfected control group: *, p<0.05; **,p<0.001; and compared with HIV-1/VSV group: #, p<0.001. For FIG. 43C,HIV-1/VSV-infected BMM were stereotactically injected i.c. into thebasal ganglia of syngeneic C57BL/6J mice. Treg or Teff (1×10⁶) wereadoptively transferred into HIVE mice 1 day postinfection. Serialsections of brain tissue that comprise the injection area were obtainedon day 7 postinfection and analyzed by immunohistochemistry for TUNELand nuclei by DAPI stain. Scale bars: 50 μm; original magnification:×400.

FIG. 44 shows Treg reduce HIV-1 viral replication in BMM. HIV-1/VSVinfected BMM were cocultured with Teff or Treg at a ratio of 3:1 (BMM vsTreg or BBB vs Teff). FIG. 44A shows supernatants from cultures ofuninfected BMM (Control; closed triangles), HIV-1/VSV-infected BMM(closed squares), HIV-1/VSV/Teff (open circles), and HIV-1/VSV/Treg(open diamonds) which were collected from day 1 to day 6 after theaddition of T cells and assessed for reverse transcriptase (RT)activity. For FIG. 44B, virally infected BMM cultured in the absence orpresence Teff or Treg or uninfected BMM (Control) were harvested at theend of day 6 and stained for expression of HIV-1 p24 and visualized bylight microscopy at ×400 original magnification. Scale bars equal 50 μm.For FIG. 44C, HIV-1 p24-positive BMM were counted and percentages ofHIV-p24-positive BMM were determined. For FIGS. 44A and 44C, means±SEMwere determined from three independent experiments. Compared withHIV-1/VSV BMM group by Student's test: #, p<0.01; ##, p<0.001.

FIG. 45 shows Treg inhibit ROS production and cytotoxicity in BMM.Anti-CD3-activated Treg or Teff were cocultured in the absence orpresence of HIV-1/VSV-infected BMM for 24 hours. After removal of Tcells, ROS production was measured as a function of H₂O₂ accumulationusing an Amplex Red assay. Uninfected BMM cultured alone served as ROSbaseline controls. For FIG. 45A, compared with uninfected BMM controls(open bar), percentage increases of ROS as a function of H₂O₂ levelswere determined for HIV-1/VSV-infected BMM cultured alone (gray bar) orcocultured in the presence of Teff (HIV-1 VSV/Teff, speckled bar), orTreg (HIV-1/VSV/Treg, black bar). For FIG. 45B, uninfected BMM culturedin the absence (left set) or presence of PMA (right set) were coculturedwithout TNF-α or T cells (open bars) in the presence of TNF-α (graybars), with TNF-α and Teff (speckled bars), or with TNF-α and Treg(black bars). For FIGS. 45A and 45B, mean±SEM were determined for threeexperimental determinations and significant differences were determinedby Student's t test; compared with control group: **, p<0.01; ***,p<0.001; compared with HIV-1/VSV group: ##, p<0.01; ###, p<0.001. ForFIG. 45C, uninfected BMM (Control) and HIV-1/VSV-infected BMM werecultured in the absence or presence of Teff or Treg for 24 hours andsupernatants were harvested. Collected supernatants were then subjectedto cytokine array blots (RayBiotech). Ovals encompass replicate blots todetect expression of IL-2 (oval 1), IL-12p40/p70 (oval 2), MCP-1 (oval3), and MCP-5 (oval 4). For FIG. 45D, densitometric analysis of cytokinearray blots were achieved by digital image analysis with NIH Image Jsoftware, and mean densities±SD were determined for replicatedeterminations. Mean densities±SD of positive controls for each array ofcontrol BMM and HIV-1/VSV-infected BMM cultured in the absence orpresence of Teff or Treg were 204.4±9.6, 206.3±6.0, 201.7±14.8, and202.5±11.9, respectively.

FIG. 46 shows Treg induce neuroprotective responses from HIV-1/VSVinfected BMM. Mouse primary neurons were exposed for 24 hours to 10% ofCM collected from uninfected BMM (Control), HIV-1/VSV-infected BMM, orHIV-1 /VSV-infected BMM cocultured with Treg or Teff. For FIG. 46A,treated primary neuronal isolates were immunostained for expression ofMAP-2 and NeuN. Images are at ×400 original magnification and the scalebars equal 50 μm (top row). Apoptotic neurons were determined by TUNELstaining showing apoptotic cells and DAPI nuclear staining. Micrographsare shown at ×200 magnification and scale bars equal 100 μm (middle andlower rows). For FIG. 46B, percentages of apoptotic neurons treated withCM from control BMM, HIV-1/VSV-infected BMM, HIV-1/VSV-infected BMMtreated with Teff, or HIV-1/VSV infected BMM treated with Treg. Meanpercentages±SEM were determined from three experiments; compared withcontrol group: ***, p<0.001; and compared with HIV-1/VS infected BMMgroup: ###, p<0.001.

FIG. 47 shows the effect of COP-1 immunization in B6 SOD1 mice. Micewere treated with PBS (closed circles and black bars), COP-1 weekly(q1wk) (open boxes and gray bars), or COP-1 every 2 weeks (q2wk) (opentriangles and white bars). FIG. 47A shows a Kaplan-Meier analysis of theproportion of surviving SOD1 Tg mice as a function of age. Cox's F-testcomparison showed groups treated with PBS vs COP-1 q1wk (p=0.0413) orCOP-1 q2wk (p=0.1151), and COP-1 q1wk vs COP-1 q2wk (p=0.1673). FIG. 47Bshows a log-normal analysis of mortality probability at 10 day intervalsfor mice treated with PBS, COP-1 q1wk, COP-1q2wk. FIG. 47C provides aKaplan-Meier plot of the proportion of surviving female SOD1 Tg mice(left panel) or male SOD1 Tg mice (right panel) as a function of ageshowing the gender effect. Cox's F-test comparison showed female micegroups treated with PBS vs COP-1 q1wk (p=0.0434) or COP-1 q2wk(p=0.2449), and COP-1 q1wk vs COP-1 q2wk (p=0.0846) and male mice groupstreated with PBS vs COP-1 q1wk (p=0.4240) or COP-1 q2wk (p=0.1615), andCOP-1 q1wk vs COP-1 q2wk (p=0.1430). FIG. 47D provides the mean age ofsurvival±SEM for 7-10 female or 5-6 male SOD1 Tg mice/group treated withPBS, COP-1 q1wk, or COP-1 q2wk. ^(a)P<0.05 compared to PBS treated micewith Bonferroni post-hoc tests. Spleen cells from B6 Tg mice treatedwith PBS, COP-1 q1wk, or COP-1 q2wk were stimulated with (FIG. 47E)Cop-1 (5 μg/ml), (FIG. 47F) Con A (2 μg/ml) or cultured in media alone.Cells were pulsed with [³H]-TdR for the final 18 hours of culture,harvested onto glass fiber filters and counted by b-scintillationspectrometry. Counts were normalized as a ratio of those obtained fromculture in media alone to generate a stimulation index for spleen cellproliferation from each animal. A stimulation index of 1 is defined byspleen cells cultured in media alone (dashed line). Means of stimulationindices (6SEM) were determined from 4-5 mice/group for (FIG. 47E)antigen-specific proliferation elicited by Cop-1 and (FIG. 47F)polyclonal T cell proliferation induced by the T cell mitogen, Con A.^(a)P<0.05, above media control (stimulation index=1, dashed line); and^(b)P<0.05 compared to weeks 4 or 8 within each treatment group.

FIG. 48 shows spleen changes in G93A-SOD1 Tg mice. FIG. 48A shows themorphology and size of spleens from B6SJL SOD1 Tg mice and Wtlittermates at 14 weeks of age (left panel) and 20 weeks of age (rightpanel). FIG. 48B shows mean spleen weights were compared between B6SJLWtlittermates and B6SJL SOD1 Tg mice at 7, 16 and 19 weeks age and betweenB6 Wt and B6 SOD1 Tg mice at 22 weeks age (n=5-9 mice/group). FIG. 48Cshows total spleen cell numbers were compared between Wt littermates andB6SJL SOD1 Tg mice at 14 and 22 weeks of age. Values are means±SEM for3-9 mice per group.

FIG. 49 shows altered spleen architecture from end stage G93A-SOD1 Tgmice. Representative photomicrographs of immunohistochemistry are shownfor expression of CD3, F4/80, Gr-1, CD19 of fresh frozen spleen sectionsfrom end stage SOD1 Tg mice and age-matched Wt controls.Photomicrographs in the left panels are from B6 Wt mice, while middleand right panels show sections from B6 SOD1 Tg and B6SJL SOD1 Tg mice,respectively. Sections are stained by immunoperoxidase for expression of(A, B, C) CD3 by T cells; (D, E, F) F4/80 by perifollicular macrophages;(G, H, I) Gr-1 immunoreactivity on myeloid cells; and (J, K, L) CD19+ onB cells. Sections are counterstained with hematoxylin.

FIG. 50 shows the comparison of spleen architecture between Wt and SOD1B6 Tg mice (22 weeks old). Mean area/follicle (FIG. 49A) and meannumbers of follicles/mm² (FIG. 50B) were determined for B6 Wt and B6SOD1 Tg mice from digital images taken at 100× magnification (4 randomfields/mouse). Densities of CD3+ T cells (FIG. 50C), F4/80+ macrophages(FIG. 49D), Gr-1+ cells (FIG. 50E), and CD19+B cells (FIG. 50F) fromconcomitantly stained sections were determined by digital image analysisfrom 100× magnifications using Image-Pro Plus software. Values aremeans6SEM for 3-6 mice per group.

FIG. 51 shows lymphocyte phenotype and function in G93A-SOD1 Tg mice.The phenotype and function of splenic lymphocytes from B6SJL SOD1 Tg andWt littermates were assessed by flow cytometric analysis (FCM) andproliferation assays. FIG. 51A shows representative dot plot for FCManalysis of CD4+ gated naive (CD44^(lo)CD62L^(hi)) and memory(CD44^(hi)CD62^(lo)) T cells from Wt (left) and SOD1 Tg (right) mice at14 weeks of age. For FIG. 50B, mean percentages (±SEM) of CD4+ naive andmemory T cells (left panel) and ratios of naive/memory CD4+ T cells(right panel) were determined for 5 Wt and 5 B6SJL SOD1 Tg mice. ForFIG. 50C, percentages of annexin-V+7ADD+ (necrotic) and annexin-V+7ADD2(apoptotic) Thy-1+T cells or CD45RB220+B cells amongst spleniclymphocytes were assessed in Wt and B6SJL SOD1 Tg mice at 14 weeks ofage. For FIG. 50D, lymphoproliferative responses of Wt littermates andB6SJL SOD1 Tg mice at 14 weeks of age were evaluated after in vitrostimulation for 3 days with anti-CD3 (1 μg/ml), anti-IgM (20 μg/ml), ormedia alone. Stimulation indices for [³H]-TdR uptake by splenocytes fromeach animal were determined from quadruplicate cultures and valuesrepresent the mean±SEM for 5 mice per group.

FIG. 52 shows the effect of total lymphocyte reconstitution on survivaland clinical scores in B6 SOD1 Tg mice. B6 SOD1 mice (20 mice/group)were treated with PBS (closed circles) or RCS (open circles) with 50×10⁶naive splenic lymphocytes. FIG. 52A provides Kaplan-Meier analysis ofthe proportion of surviving SOD1 Tg mice as a function of age. P=0.2035by Cox's F-test for comparison of PBS and RCS groups. FIG. 52B providesmean age of survival±SEM for 20 mice/group treated with PBS (black bar,149.3±7.5 days) or reconstituted with naive lymphocytes (RCS) (whitebar, 151.5±7.5 days). Comparison of treatment groups indicated p=0.315by ANOVA. FIG. 52C provides mean clinical scores (±SEM) of PBS- orRCS-treated SOD1 Tg mice as a function of age in weeks. *P<0.05 comparedto PBS treated group by factorial ANOVA and Fisher's LSD post-hoc testsof treatment and age. FIG. 52D provides Kaplan-Meier analysis of age andcumulative proportion of SOD1 Tg mice reaching onset of disease(clinical score=3). P=0.0012 by Cox's F-test comparison of reconstitutedmice to those treated with PBS. FIG. 52E provides Kaplan-Meier analysisof age and proportion of SOD1 Tg reaching late disease stage (clinicalscore=1). P=0.0191 by Cox's F-test comparison of reconstituted mice tothose treated with PBS. FIG. 52F provides Kaplan-Meier analysis of thecumulative proportion of SOD1 Tg mice surviving after the time ofdisease onset (clinical score=3). P=0.2021 by Cox's F test comparison ofRCS and those mice treated with PBS.

FIG. 53 shows the effect of Treg and Teff on survival, clinical scoresand weight loss in B6 SOD1 Tg mice. B6 G93A-SOD1 mice (14-15 mice/group)were treated with PBS (closed circles), 1×10⁶ activated Treg (openboxes), or 1×10⁶ activated Teff (open triangles) at 7, 13, and 19 weeksof age. FIG. 53A provides Kaplan-Meier analysis of the proportion ofsurviving SOD1 Tg mice as a function of age. Cox's F-test comparison ofgroups treated with PBS vs Treg (p=0.0054) or Teff (p=0.0002), and Tregvs Teff (p=0.2505). FIG. 53B provides clinical scores of SOD1 Tg mice asa function of age in weeks. *P<0.05 compared to PBS treated group ateach time point by factorial ANOVA and Fisher's LSD post-hoc tests. FIG.53C provides Kaplan-Meier analysis for age and proportion of SOD1 Tgreaching late disease stage (clinical score=1). Cox's F-test comparisonof groups treated with PBS vs Treg (p=0.006) or Teff (p=0.0003), andTreg vs Teff (p=0.1883). FIG. 53D provides Kaplan-Meier analysis of ageand cumulative proportion of SOD1 Tg mice reaching onset of disease(clinical score=3). Cox's F test comparison of groups treated with PBSvs Treg (p=0.002) or Teff (p=0.4215), and Treg vs Teff (p=0.0014). FIG.53E provides Kaplan-Meier analysis of the cumulative proportion of SOD1Tg mice and the time after disease onset (clinical score=3) to reachlate stage (clinical score=1). Cox's F test comparison of groups treatedwith PBS vs Treg (p=0.2716) or Teff (p=0.0098), and Treg vs Teff(p=0.0055). FIG. 53F provides Kaplan-Meier analysis of the age and thecumulative proportion of SOD1 Tg mice that exhibited a reduction ofmaximum body weight ≧10%. Cox's F-test comparison of groups treated withPBS vs Treg (p=0.2131) or Teff (p=0.003), and Treg vs Teff (p=0.0807).

FIG. 54 shoes the effect of Treg and Teff on mean age of survival,clinical scores and weight loss in B6 G93A-SOD1 Tg mice. B6 G93A-SOD1mice were treated with PBS (black bars), 1×10⁶ activated Treg (graybars), or 1×10⁶ activated Teff (white bars) at 7, 13, and 19 weeks ofage. FIG. 54A provides mean age of survival±SEM for 14-15 SOD1 Tgmice/group treated with PBS (152.7±2.0 days), Treg (165.8±5.0 days), orTeff (170.1±3.4 days). ^(a)P<0.04 compared to PBS-treated mice by ANOVAand Bonferroni post-hoc tests. FIG. 54B provides mean age±SEM for SOD1Tgmice reaching late stage (clinical score=1) after treatment with PBS(136.9±3.1 days), Treg (153.5±5.8 days), or Teff (160.1±4.3 days).^(a)P<0.04 compared to PBS control group by ANOVA and Bonferronipost-hoc tests. FIG. 54C provides mean age±SEM for SOD1 Tg mice atdisease onset (clinical score=3) after treatment with PBS (77.7±1.3days), Treg (89.8±2.7 days), or Teff (75.7±1.9 days). ^(a)P=0.0003compared to PBS control group by ANOVA and Bonferroni post-hoc tests.FIG. 54D provides mean age±SEM for SOD1 Tg mice that exhibit a reductionof maximum body weight ≧10% after treatment with PBS (147.2±2.5 days),Treg (152.8±3.7 days), or Teff (159.7±4.0 days). ^(a)P<0.04 compared toPBS control group by ANOVA and Bonferroni post-hoc tests.

FIG. 55 shows the effect of Treg and Teff on motor function in B6G93A-SOD1 Tg mice. B6 G93A-SOD1 mice (14-15 mice/group) were treated at7, 13, and 19 weeks of age with PBS (closed circles and black bars),1×10⁶ activated Treg (open boxes and gray bars), or 1×10⁶ activated Teff(open triangles and white bars). FIG. 55A provides Kaplan-Meier analysisof the age and the cumulative proportion of SOD1 Tg mice that exhibiteda ≧75% reduction of overall rotarod performance (ORP). Cox's F-testcomparison of groups treated with PBS vs Treg (p=0.0377) or Teff(p=0.0084), and Tregs vs Teffs (p=0.27). FIG. 55B provides mean age±SEMfor 14-15 SOD1 Tg mice/group at which mice exhibited a $75% reduction inORP after treatment with PBS (142.1±2.8 days), Treg (154.0±5.6 days), orTeff (157.5±4.4 days). ^(a)P<0.05 compared to PBS treated mice by ANOVAand Bonferroni post-hoc tests. FIG. 55C provides Kaplan-Meier analysisof ages and cumulative proportion of SOD1 Tg mice that exhibited ≧25%reduction in ORP. Cox's F-test comparison of groups treated with PBS vsTreg (p=0.0054) or Teff (p=0.0020), and Treg vs Teff (p=0.20). FIG. 55Dprovides mean age±SEM for 14-15 SOD1 Tg mice/group at which miceexhibited ≧25% reduction in ORP after treatment with PBS (117.7±3.5days), Treg (136.5±6.2 days), or Teff (143.7±6.1 days). ^(a)P<0.0025compared to PBS treated mice by ANOVA and Bonferroni post-hoc tests.FIG. 55E provides Kaplan-Meier analysis for age and proportion of SOD1Tg mice that exhibited ≧75% reduction of maximum Paw Grip Endurance(PaGE) after treatment with PBS, Treg, or Teff. Cox's F test comparisonof groups treated with PBS vs Treg (p=0.001) or Teff (p=0.0003), andTreg vs Teff (p=0.14). FIG. 55F provides mean age±SEM for SOD1Tg micethat exhibited ≧75% reduction of maximum PaGE after treatment with PBS(94.5±5.6 days), Treg (131.1±4.2 days), or Teff (137.2±4.5 days).^(a)P<0.0001 compared to PBS control group by ANOVA and Bonferronipost-hoc tests. FIG. 55G provides Kaplan-Meier analysis for age (days)and proportion of SOD1 Tg mice that exhibited $25% reduction of maximumPaGE after treatment with PBS, Treg, or Teff. Cox's F test comparison ofgroups treated with PBS vs Treg (p<0.0001) or Teff (p=0.0033), and Tregvs Teff (p=0.42). FIG. 55G provides mean age±SEM at which SOD1 Tg miceexhibited at least 25% reduction of maximum PaGE after treatment withPBS (68.5±3.6 days), Treg (119.1±7.2 days), or Teff (108.7±11.3 days).^(a)P<0.002 compared to PBS control group by ANOVA and Dunnett'spost-hoc tests.

DETAILED DESCRIPTION OF THE INVENTION

Direct evidence is provided herein that the adaptive immune system canexacerbate dopaminergic neuronal loss in animal models of Parkinson'sdisease. Nitrated proteins that drain from the CNS into lymphaticsinduce macrophage activation and T cell responses. Moreover, it is shownthat these T cell responses to N-α-Syn elicit profoundneurodegeneration. Cell damage resulting from N-α-Syn is not limited tobrain cells as it affects both T cell function and numbers and canaffect regulatory T cell subsets. The data, taken together, indicatesthat an exacerbated immune response induced by N-α-Syn plays a role inPD pathogenesis.

It is proposed herein that modified “self” epitopes as neo-epitopes,including 3-nitrotyrosine (NT) modifications within α-Syn, can bypass orbreak immunological tolerance (Ohmori et al. (2005) Autoimmun. Rev., 4:224-229; Mevorach et al. (1998) J. Exp. Med., 188:387-392;Casciola-Rosen et al. (1997) J. Exp. Med., 185:71-79; Amoura et al.(1999) Arthritis Rheum., 42:833-843; Burkhardt et al. (2001) TrendsImmunol., 22:291-293; Utz et al. (1998) Arthritis Rheum., 41:1152-1160;Doyle et al. (2001) Trends Immunol., 22: 443-449) and activateperipheral leukocytes in draining lymphoid tissue. In keeping with this,NT-modifications incorporated into self-peptides were sufficient toevade immunological tolerance as was previously reported (Bimboim et al.(2003) J. Immunol., 171:528-532). The recruitment of activated T cells,specific for disease-associated protein modifications in α-Syn, canpromote a toxic microglial phenotype. The role of the adaptive immunesystem is becoming increasingly important in “non-autoimmune” diseasesof the CNS (Gendelman, H. E. (2002) J. Neurovirol., 8:474-479). Researchin traumatic and neurodegenerative models have suggested aneuroprotective role for T and B cells within the CNS and thatmanipulation of the peripheral immune system can affectneurodegeneration (Kipnis et al. (2002) J. Neuroimmunol., 130:78-85).Other studies demonstrated that immunization of mice with glatirameracetate generate T cells that recognize myelin basic protein (T_(MBP)),secrete interleukin (IL)-10, IL-4, and transforming growth factor(TGF)-β, and confer protection against1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-inducedneurodegeneration presumably by suppression of microglial activation(Benner, et al. (2004) Proc. Natl. Acad. Sci., 101:9435-9440).Antibodies generated through active immunization of human α-Syntransgenic mice with purified human α-Syn protein reduced α-Synaggregation in cell bodies and terminals, and was associated withprotection of dopaminergic nerve terminals (Masliah et al. (2005)Neuron, 46:857-868). The conclusions were that anti-α-Syn antibodiestarget the aggregated protein to lysosomal pathways for degradation andthat the strategy could be applied for treatment of human disease. Thatwork was conducted however in an animal model of PD that lacks aneuroinflammatory component. As such, the study did not address thecellular arm of the immune system, which likely requires cytokine andchemokine gradients for efficient cell entry into diseased regions.Nevertheless, other work supports the potential importance for adaptiveimmunity and for immune-based strategies for the treatment of PD(Masliah et al. (2005) Neuron, 46:857-868).

Herein, it is reported that NT-modified CNS antigens drain to the deepcervical lymph nodes (CLN) of mice following exposure to MPTP. Moreover,antigen-presenting cells (APC) within CLN increase surface expression ofmajor histocompatibility complex (MHC) class II, initiating themolecular machinery necessary for efficient antigen presentation. Thedifferential outcome on the susceptibility to MPTP-induced dopaminergicneurodegeneration amongst WT and severe combined immunodeficient (SCID)mice suggest a functional link of the adaptive immune system toMPTP-induced neurotoxicity. It is further demonstrated in mice of twodisparate haplotypes, that adoptive transfer of T cells from syngeneicWT donors immunized with nitrated α-Syn (N-α-Syn) prolongs MPTP-induceddopaminergic neuronal loss and hence warrants caution against the use ofN-α-Syn or self-proteins that are prone to nitrate modifications forvaccine-based PD therapies.

It is also shown herein that N-α-syn immunization elicits adaptiveimmune responses, to novel antigenic epitopes, that exacerbatenigrostriatal degeneration in the1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model ofParkinson's disease (PD). Such neuroimmune degenerative activities, inmost significant measure, are Th17 mediated and accelerated throughnumbers of dysfunctional CD4+ CD25+ regulatory T cells (Treg). These arecontained within N-α-syn T cell mixtures. Significantly, Tregreconstitution by vasoactive intestinal peptide (VIP) reversesneurodegeneration by induction of a robust neuroprotective T cellresponse. These attenuate nigrostriatal destruction by N-α-syn T cells.Combinations of adoptively transferred N-α-syn and VIP immunocytes toMPTP mice produce nigrostriatal protection associated with reducedmicroglial inflammatory responses. Taken together, these resultsdemonstrate that Treg halts N-α-syn neurodestructive immunity providinga sound rationale for PD immunization strategies.

In accordance with one aspect of the instant invention, methods oftreating a central nervous system disease or disorder are provided. In apreferred embodiment, the central nervous system disease or disorder ischaracterized by the presence of an abnormal protein(s) (e.g., a mutantprotein and/or over-expressed protein (including inappropriateexpression at a particular site)). In a particular embodiment, themethods comprise administering to a subject in need thereof: 1) at leastone immunogen capable of inducing a humoral immune response against theabnormal protein(s) and 2) at least one adjuvant that stimulatesfunctional regulatory T cells. In a preferred embodiment, the humoralimmune response causes the abnormal protein to be substantially clearedand eliminated from the site where it is located within the centralnervous system. The immunogen and adjuvant may be contained in the samecomposition or in separate compositions. When the compositions areadministered separately, the compositions may be administeredsimultaneously or sequentially (e.g., the adjuvant may be administeredfirst and then the immunogen, the immunogen may be administered firstand then the adjuvant, or multiple administrations of each component maybe used in any order).

While the above methods are described as treating a central nervoussystem diseases or disorders, the methods of the instant invention alsoencompass the prevention of a central nervous system disease ordisorder. In a particular embodiment, the methods of the instantinvention delay or inhibit the onset of the central nervous systemdisease or disorder and/or symptoms associated therewith. For example,the compositions of the instant invention may be administered to ahealthy individual, particularly one at risk for a central nervousdisease or disorder.

The methods of the instant invention may further comprise administeringother therapies which are beneficial to the treatment of the particularcentral nervous system disease or disorder. The methods of the instantinvention may also further comprise the step of monitoring the subjectfor the central nervous system disease or disorder after theadministration of the compositions of the instant invention. Forexample, the subject may be monitored at least once, at least twice, atleast three times or more after treatment. The monitoring may beperformed over the course of weeks, months, and/or years. The centralnervous system disease or disorder may be monitored through, forexample, biological (clinical) diagnosis and/or monitoring of thesymptoms associated with the central nervous system disease or disorder.

In accordance with another aspect of the instant invention, compositionsfor the treatment/prevention of a central nervous system disease ordisorder are provided. In one embodiment, the composition comprises 1)at least one immunogen capable of inducing a humoral immune responseagainst an abnormal protein(s) and 2) at least one adjuvant thatstimulates the production of regulatory T cells. In a particularembodiment, the instant invention encompasses a kit comprising at leasttwo compositions: wherein at least one composition comprises the atleast immunogen and, optionally, at least one pharmaceuticallyacceptable carrier; and at least one other composition comprises the atleast one adjuvant and, optionally, at least one pharmaceuticallyacceptable carrier.

The central nervous disease or disorder of the instant invention can beany central nervous system disease or disorder. In a preferredembodiment, the central nervous system disease or disorder tocharacterized by at least one disease specific protein against which animmune response is desirable. Examples of central nervous systemdiseases and disorders include, without limitation, multi-infarctdementia, stroke, Pick's Disease, frontal lobe degeneration,corticobasal degeneration, multiple system atrophy, progressivesupranuclear palsy, Creutzfeldt-Jakob disease, lewy body disease,neuroinflammatory disease, Parkinson's Disease, Alzheimer's Disease,amyotrophic lateral sclerosis, neuroAIDS, Chron's Disease, andHuntington's Disease. In a particular embodiment of the instantinvention, the central nervous system disease is selected from a groupconsisting of Parkinson's Disease, Alzheimer's Disease, amyotrophiclateral sclerosis, neuroAIDS, Chron's Disease, and Huntington's Disease.

In a particular embodiment, the adjuvant induces a T cell phenotypicswitch from pro-inflammatory (TH1 and TH17) to anti-inflammatory andregulatory (TH2, Treg, and Tr1). In one embodiment, the adjuvant of themethods and compositions of the instant invention is selected from thegroup consisting of glatiramer acetate (Cop-1, Copaxone®), vasoactiveintestinal peptide, vitamin D (1 alpha, 25-dihydroxyvitamin D3),granulocyte macrophages colony stimulating factor, and transforminggrowth factor beta. In a particular embodiment, the adjuvant isvasoactive intestinal peptide (particularly human, but the instantinvention encompasses VIP from other species).

As stated hereinabove, the immune response (e.g., humoral immuneresponse) induced by the administration of a composition of the instantinvention to a subject causes the abnormal protein (includingpolypeptides and peptides) which characterizes the central nervousdisease or disorder to be substantially reduced (preferably eliminated)from the site of abnormal expression (e.g., within the central nervoussystem). In a particular embodiment, the immunogen comprises at leastone antigenic epitope of the abnormal protein. In yet anotherembodiment, the immunogen is the abnormal protein. Immunogens include,without limitation, alpha synuclein (preferably nitrated alphasynuclein), amyloid beta (particularly the amyloid beta associated withAlzheimer's (e.g., amyloid beta 42)), and superoxide dismutase(preferably, superoxide dismutase 1). In a particular embodiment, theimmunogen is alpha synuclein when the central nervous disease isParkinson's Disease; the immunogen is amyloid beta when the centralnervous disease is Alzheimer's Disease; the immunogen is superoxidedismutase when the central nervous system disease is amyotrophic lateralsclerosis; and the immunogen is HIV (e.g., attenuated/dead) and/orenvelope glycoprotein (e.g., gp120) when the central nervous systemdisease is neuroAIDS.

The alpha synuclein is preferably nitrated. The alpha synuclein may befrom any species, particularly human. An example of an amino acidsequence of alpha synuclein is SEQ ID NO: 13 (see also GenBank AccessionNo. P37840). An alpha synuclein amino acid sequence may have 75%, 80%,85%, 90%, 95%, 97%, or 99% homology with any of the alpha synucleinsequences provided herein. In a particular embodiment, a fragment ofalpha synuclein is used. In a particular embodiment, the fragment is theC-terminal 40 amino acids of alpha synuclein (e.g., amino acids 101-140of SEQ ID NO: 13). In another embodiment, the fragment is a peptideconsisting of anywhere from the C-terminal 20 amino acids to theC-terminal 70 amino acids, particularly the from the C-terminal 30 aminoacids to the C-terminal 50 amino acids. In a particular embodiment, thefragment is the C-terminal 40 amino acids plus or minus 1, 2, 3, 4, or5, amino acids. The fragments may optionally have an N-terminalmethionine added. The fragments may have 75%, 80%, 85%, 90%, 95%, 97%,or 99% homology with the above alpha synuclein sequences. As statedhereinabove, the alpha synuclein is preferably nitrated. The alphasynuclein of fragment thereof may comprise at least one, at least two,at least three, at least four, at least five or all of the tyrosinesnitrated into nitrotyrosines.

The compositions described herein will generally be administered to apatient as a pharmaceutical preparation. The term “patient” or“subject”, as used herein, refers to human or animal subjects. Thecompositions of the instant invention may be employed therapeutically,under the guidance of a physician.

The compositions of the instant invention may be conveniently formulatedfor administration with any pharmaceutically acceptable carrier(s). Forexample, the complexes may be formulated with an acceptable medium suchas water, buffered saline, ethanol, polyol (for example, glycerol,propylene glycol, liquid polyethylene glycol and the like), dimethylsulfoxide (DMSO), oils, detergents, suspending agents or suitablemixtures thereof. The concentration of the active agents in the chosenmedium may be varied and the medium may be chosen based on the desiredroute of administration of the pharmaceutical preparation. Exceptinsofar as any conventional media or agent is incompatible with thepolymer-therapeutic agent complexes to be administered, its use in thepharmaceutical preparation is contemplated.

The dose and dosage regimen of the compositions according to theinvention that are suitable for administration to a particular patientmay be determined by a physician considering the patient's age, sex,weight, general medical condition, and the specific condition for whichthe polymer-therapeutic agent complex is being administered and theseverity thereof. The physician may also take into account the route ofadministration, the pharmaceutical carrier, and the particular agent'sbiological activity. Selection of a suitable pharmaceutical preparationwill also depend upon the mode of administration chosen. For example,the compositions of the invention may be administered by directinjection to a desired site. In this instance, a pharmaceuticalpreparation comprises the active agents of the instant inventiondispersed in a medium that is compatible with the site of injection. Thecompositions of the instant invention may be administered by any method.For example, the compositions can be administered, without limitationparenterally, subcutaneously, orally, topically, pulmonarily, rectally,vaginally, ocularly, intravenously, intraperitoneally, intracranial,intrathecally, intracerbrally, epidurally, intramuscularly,intradermally, or intracarotidly. In a particular embodiment, thecompositions are administered intravenously, subcutaneously, or orallyor by direct injection. Pharmaceutical preparations for injection areknown in the art. If injection is selected as a method for administeringthe compositions, steps must be taken to ensure that sufficient amountsof the molecules reach their target cells to exert a biological effect.Dosage forms for oral administration include, without limitation,tablets (e.g., coated and uncoated, chewable), gelatin capsules (e.g.,soft or hard), lozenges, troches, solutions, emulsions, suspensions,syrups, elixirs, powders/granules (e.g., reconstitutable or dispersible)gums, and effervescent tablets. Dosage forms for parenteraladministration include, without limitation, solutions, emulsions,suspensions, dispersions and powders/granules for reconstitution. Dosageforms for topical administration include, without limitation, creams,gels, ointments, salves, patches and transdermal delivery systems.

A pharmaceutical preparation of the invention may be formulated indosage unit form for ease of administration and uniformity of dosage.Dosage unit form, as used herein, refers to a physically discrete unitof the pharmaceutical preparation appropriate for the patient undergoingtreatment. Each dosage should contain a quantity of active ingredientcalculated to produce the desired effect in association with theselected pharmaceutical carrier. Procedures for determining theappropriate dosage unit are well known to those skilled in the art.

Dosage units may be proportionately increased or decreased based on theweight of the patient. Appropriate concentrations for alleviation of aparticular pathological condition may be determined by dosageconcentration curve calculations, as known in the art.

In accordance with the present invention, the appropriate dosage unitfor the administration of the composition may be determined byevaluating the toxicity of the molecules or cells in animal models.Various concentrations of active agents in pharmaceutical preparationsmay be administered to mice or other animal models, and the minimal andmaximal dosages may be determined based on the beneficial results andside effects observed as a result of the treatment. Appropriate dosageunit may also be determined by assessing the efficacy of the treatmentin combination with other standard drugs. The dosage units of thecompositions of the instant invention may be determined individually orin combination with each treatment according to the effect detected.

The pharmaceutical preparation of the instant invention may beadministered at appropriate intervals (e.g., at least one booster), forexample, once every 2-4 days, once a week, or once every 2-6 of weeksuntil the pathological symptoms are reduced or alleviated, after whichthe dosage may be reduced to a maintenance level or eliminated. Theappropriate interval in a particular case would normally depend on thecondition of the patient. In a particular embodiment, the composition isadministered to the body in an isotonic solution at physiological pH7.4. However, the complexes can be prepared before administration at apH below or above pH 7.4.

DEFINITIONS

The term “treat” as used herein refers to any type of treatment thatimparts a benefit to a patient afflicted with a disease, includingimprovement in the condition of the patient (e.g., in one or moresymptoms), delay in the progression of the condition, etc.

A “therapeutically effective amount” of a compound or a pharmaceuticalcomposition refers to an amount effective to prevent, inhibit, treat, orlessen the symptoms of a particular disorder or disease. The treatmentof a central nervous disease or disorder herein may refer to curing,relieving, and/or preventing the central nervous system disease ordisorder, a symptom(s) of it, or the predisposition towards it.

“Pharmaceutically acceptable” indicates approval by a regulatory agencyof the Federal or a state government or listed in the U.S. Pharmacopeiaor other generally recognized pharmacopeia for use in animals, and moreparticularly in humans.

A “carrier” refers to, for example, a diluent, adjuvant, preservative(e.g., Thimersol, benzyl alcohol), anti-oxidant (e.g., ascorbic acid,sodium metabisulfite), solubilizer (e.g., Tween 80, Polysorbate 80),emulsifier, buffer (e.g., Tris HCl, acetate, phosphate), bulkingsubstance (e.g., lactose, mannitol), excipient, auxilliary agent,filler, disintegrant, lubricating agent, binder, stabilizer,preservative or vehicle with which an active agent of the presentinvention is administered. Pharmaceutically acceptable carriers can besterile liquids, such as water and oils, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, sesame oil and the like. Water or aqueous saline solutionsand aqueous dextrose and glycerol solutions are preferably employed ascarriers, particularly for injectable solutions. The compositions can beincorporated into particulate preparations of polymeric compounds suchas polylactic acid, polyglycolic acid, etc., or into liposomes ormicelles. Such compositions may influence the physical state, stability,rate of in vivo release, and rate of in vivo clearance of components ofa pharmaceutical composition of the present invention. Thepharmaceutical composition of the present invention can be prepared, forexample, in liquid form, or can be in dried powder form (e.g.,lyophilized). Suitable pharmaceutical carriers are described in“Remington's Pharmaceutical Sciences” by E. W. Martin (Mack PublishingCo., Easton, Pa.); Gennaro, A. R., Remington: The Science and Practiceof Pharmacy, 20th Edition, (Lippincott, Williams and Wilkins), 2000;Liberman, et al., Eds., Pharmaceutical Dosage Forms, Marcel Decker, NewYork, N.Y., 1980; and Kibbe, et al., Eds., Handbook of PharmaceuticalExcipients (3rd Ed.), American Pharmaceutical Association, Washington,1999.

The term “neuroAIDS” (also referred to as HIV-associated neurocognitivedisorders), as used herein, encompasses those novel neurologic disorderswhich are a primary consequence of damage to the central nervous systemby HIV. The clinical syndromes identified include sensory neuropathy,myelopathy, HIV dementia, and cognitive/motor disorder.

The term “isolated” may refer to a compound or complex that has beensufficiently separated from other compounds with which it wouldnaturally be associated. “Isolated” is not meant to exclude artificialor synthetic mixtures with other compounds or materials, or the presenceof impurities that do not interfere with fundamental activity or ensuingassays, and that may be present, for example, due to incompletepurification, or the addition of stabilizers.

An “immunogen” refers to a compound comprising a peptide, polypeptide orprotein which is “immunogenic,” i.e., capable of eliciting, augmentingor boosting an immune response (e.g., cellular and/or humoral). Theimmunogen can be recombinantly produced. An immunogen comprises at leastone antigenic determinant or epitope.

As used herein, “regulatory T cells” are CD4+ CD25+ cells that exhibitimmunoinhibitory properties.

The following examples are provided to illustrate certain embodiments ofthe invention. They are not intended to limit the invention in any way.

Example 1 Materials and Methods Animals

Male 6-7 week old, WT C57BL/6J (stock 000664, denoted as B6) (H-2^(b)),B6.CB17-Prkdc^(scid)/SzJ (stock 001913, herein denoted as SCID)(H-2^(b)) and B10.BR-H2k H2-T18^(a)/SgSnJ (stock 000465, herein denotedas B10.BR) (H-2^(k)) mice were purchased from Jackson Laboratories (BarHarbor, Me.). All animal procedures were in accordance with NationalInstitutes of Health (NIH) guidelines and approved by the InstitutionalAnimal Care and Use Committee (IACUC) of the University of NebraskaMedical Center (UNMC).

MPTP Intoxication

For chronic intoxication, B6 mice received 5 intraperitoneal (i.p.)injections at 24 hour intervals for 5 days of either vehicle (PBS, 10ml/kg) or MPTP-HCl (30 mg/kg of free base in PBS) (Sigma-Aldrich, St.Louis, Mo.). For acute intoxication, mice received 4 i.p. injections,one every 2 hours, of either vehicle (PBS, 10 ml/kg) or MPTP-HCl (18mg/kg of free base in PBS for B10.BR mice, 14 or 18 mg/kg for B6 mice).At selected time points following MPTP intoxication, mice weresacrificed and brains processed for subsequent analyses. MPTP handlingand safety measures were in accordance with published guidelines(Przedborski et al. (2001) J. Neurochem., 76:1265-1274).

Immunohistochemistry

At the time points indicated following MPTP intoxication, mice weretranscardially perfused with 4% paraformaldehyde (PFA) in 0.1 M PBSusing 0.9% saline as vascular rinse. Brains were post-fixed in 4% PFAovernight, kept in 30% sucrose for 2 days, snap frozen, embedded in OCTcompound, and 30 mm sections cut on a cryostat (CM1900, Leica,Bannockburn, Ill.). The sections were collected in PBS and processedfree-floating. Primary antibodies used for immunohistochemistry includesrabbit anti-TH antibody (1:2000; Calbiochem/EMD Biosciences, Inc., SanDiego, Calif.), rat anti-mouse CD11b or Mac-1 (1:1,000; Serotec,Raleigh, N.C.), rat anti-CD3 (1:800; BD Pharmingen, San Diego, Calif.,)rat anti-CD4 (BD Pharmingen), and rat anti-CD8 (BD Pharmingen).Immunostaining was visualized using diaminobenzidine (Sigma-Aldrich) asthe chromogen and mounted on slides. TH, CD3-, CD4- andCD8-immunostained brain sections were counterstained with thionin(Sigma-Aldrich) as previously described (Benner et al. (2004) Proc.Natl. Acad. Sci., 101:9435-9440; Wu et al. (2003) Proc. Natl. Acad.Sci., 100:6145-6150). Fluoro-Jade C (Chemicon International, Inc.,Temecula, Calif.) was used to stain degenerating neurons (Schmued et al.(2005) Brain Res., 1035:24-31) and was detected as green fluorescence byfluorescence microscopy with FITC filter (Eclipse E800, Nikon, Inc.,Melville, N.Y.).

Stereology of TH-Positive Neurons

Total numbers of Niss1- and TH-stained neurons throughout the entireSNpc were counted stereologically in a blinded fashion with StereoInvestigator software (MicroBrightfield, Williston, Vt.) using theOptical Fractionator probe module as previously described (Benner et al.(2004) Proc. Natl. Acad. Sci., 101:9435-9440).

Cloning α-Syn and 4YSyn

Total RNA from adult C57BL/6 mouse brain was extracted using TRIzol®reagent (Invitrogen, Carlsbad, Calif.) according to the manufacturer'sinstructions. The full-length mouse α-Syn gene (504 bp) and 120 bplength encoding the C-terminal portion (4YSyn) was amplified by reversetranscriptase-polymerase chain reaction (RT-PCR) using Platinum® Taq DNAPolymerase High Fidelity (Invitrogen). The 5′ primer was designed tointroduce a Nde1 site at position 1. This fragment was blunt cloned intothe pZero-1 (Invitrogen) Eco RV site using standard cloning procedures.Transformed cells were plated on low salt agar containing Zeocyn and 3mM IPTG. Colonies were screened using colony PCR with α-Syn primers.Colonies containing the full-length mouse α-Syn gene or the 39 fragmentencoding 4YSyn were grown overnight and plasmid DNA was isolated usingstandard mini-prep (Invitrogen). The gene was digested out of pZero withNde1 and Xho1 and subcloned into the Nde 1 and Xho1 sites in the pET-28aprokaryotic expression vector using DH5-α cells. Colonies were screenedusing colony PCR. Purified plasmids were submitted to the UNMC corefacility for sequence confirmation. Plasmids containing the completesequence were transformed into BL-21 E. coli cells for expression.Frozen glycerol stocks were maintained at −80° C.

4YSyn Expression

Glycerol stocks were streaked on Luria-Bertani (LB) agar platecontaining 30 μg/ml kanamycin. A single colony was inoculated into LBbroth containing 30 μg/ml kanamycin, grown for 8 hours, and stored at 4°C. until the following day. The starter culture was diluted 1:100 intofresh liquid medium containing 30 μg/ml kanamycin and allowed to grow toan OD₆₀₀=0.6. Expression of recombinant protein was induced by theaddition of 3 mM IPTG with continued incubation for 3 hours at 37° C.Following induction, cells were centrifuged, weighed, and stored at −80°C. until purification protocol was resumed. >90% of detectable 4YSyn wasfound in the soluble fraction.

Protein Purification and Nitration

Cell lysis was performed with BugBuster® reagent (Novagen/EMDBiosciences, Inc., San Diego, Calif.) at 5 ml/g cells with addition ofEDTA-free protease inhibitor cocktail (Calbiochem). Benzonase nuclease(Novagen) was added to reduce viscosity during lysis followingmanufacturer's instructions. Insoluble cell debris was removed bycentrifugation at 16,000×g for 20 minutes at 4° C. The soluble fractionwas directly subjected to column affinity chromatography and was carriedout in the following steps: His-tagged protein was bound to Ni-NTAHis-Bind Resin (Novagen) in Bug Buster reagent with the addition ofimidazole (10 mM). The column was washed first with 50 mM NaH₂PO₄/300 mMNaCl/20 mM imidazole, pH 8.0 and then with 50 mM NaH₂PO₄/300 mM NaCl/35mM imidazole, pH 8.0. Elution was carried out 50 mM NaH₂PO₄/300 mMNaCl/250 mM imidazole pH 8.0. Samples were separated by SDS-PAGE andstained with Brilliant Blue G-Colloidal Coomassie stain (Invitrogen) toconfirm purity of the eluted fraction. Full-length α-Syn and 4YSyn werevisualized by silver stain (Silver Xpress, Invitrogen). Purifiedfull-length α-Syn was dialyzed in 50 mM NaH₂PO₄ buffer. Thrombincleavage was carried out using biotinylated thrombin cleavage capturekit (Novagen) following manufacturer's instructions. Cleaved His-tagswere removed with Ni-NTA resin. His-tag free full-length α-Syn andHis-tagged 4YSyn (unable to remove the His-tag) were dialyzed againstwater for 24-48 hours with multiple water changes, lyophilized, andweighed. Endotoxin was removed by polymyxin B agarose beads followingmanufacturer's instructions (Sigma-Aldrich) and tested for residualendotoxin by Limulus amebocyte lysate (LAL) assay (E-Toxate,Sigma-Aldrich). Recombinant α-Syn-derived proteins were endotoxin-freeas all batches of purified proteins utilized tested below the limit ofdetection for endotoxin by LAL (<0.05 endotoxin units, EU).

Lyophilized protein was resuspended (2 mg protein/ml) in 50 mM NaH₂PO₄buffer containing 5 mM FeCl₃ as a Lewis acid. Peroxynitrite (UpstateBiotechnology, Inc. Lake Placid, N.Y.) was added dropwise to protein toachieve a 5 M excess while vigorously mixing the reaction mixture.Nitrated protein was dialyzed against water for 48 hours using multiplewater exchanges, lyophilized, and stored at −80° C.

MALDI-TOF Mass Spectrometry

MALDI-TOF mass spectrometric analysis was performed using a Voyager DEPro mass analyzer (Applied Biosystems, Framingham, Mass.), which wasexternally calibrated prior to each assay. Data acquisition wasperformed using 500 laser shots. The MS scan range was set from 500 to20,000 m/z. Saturated cyanohydroxycinnamic acid (Sigma-Aldrich) was usedas matrix in these assays and samples were manually spotted onto MALDItargets.

Enzyme-Linked immunosorbant Assay (ELISA)

Individual wells of Immunolon II ELISA plates (Thermo Electron Corp.,Waltham, Mass.) were coated with 100 μl/well of native 4YSyn or N-4YSynat 1 μg/ml PBS, pH 8.5. Plates were incubated for 2 hours at 37° C. andwashed with 0.5% Tween20/PBS, pH 7.2 (PBS-T). Nonspecific binding wasblocked by the addition of 1% bovine serum albumin in PBS, pH 7.2(PBS-BSA) and incubation at 37° C. for 1 hour. Plates were washed withPBS-T, 100 μl of 2-fold serial dilutions (from an initial 1:50 dilutionin PBS-BSA) of serum samples from MPTP- or PBS-treated mice were addedto each well, and incubated at 37° C. for 1 hour. Plates were washedwith PBS-T and 100 μl/well of a 1:5000 dilution of horseradishperoxidase (HRP)-conjugated anti-mouse IgG (SouthernBiotech, Birmingham,Ala.) was added. Plates were incubated at 37° C. for 1 hour, washed withPBS-T, and reacted with 0.012% H₂O₂ and 2.2 mM o-phenylenediaminedihydrochloride (Sigma-Aldrich) in 100 μl of 0.1 M phosphate-citratebuffer, pH 5.0. The reaction was stopped with the addition of 2 N H₂SO₄,read at 490 nm on a microplate reader (Vmax® Kinetic Microplate Reader,Molecular Devices Corporation, Sunnyvale, Calif.), and acquired dataanalyzed with interfacing SoftMax® Pro software (Molecular Devices).Serum IgG concentrations were quantified from a standard curve preparedfrom known concentrations of mouse IgG (SouthernBiotech).

Immunization and Immune Cell Adoptive Transfers

B10.BR(H-2^(K)) mice were immunized with PBS, 50 μg of 4YSyn or N-4YSynemulsified in an equal volume of CFA containing 1 mg/ml Mycobacteriumtuberculosis (Sigma-Aldrich). B6 (H-2b) mice were immunized with PBS, 10μg of 4YSyn or N-4YSyn with or without CFA. While immunization withadjuvant were administered subcutaneous (s.c.) on either side of thetail base, s.c. injections without adjuvant were given at 5 differentsites. Fourteen days after primary immunization, mice were boosted withtheir respective antigens. CFA recipient mice were boosted with theirrespective antigens emulsified in IFA (Sigma-Aldrich). Five daysfollowing their final immunizations, donor mice were sacrificed andsingle cell suspensions were prepared from the spleen and draining lymphnodes after lysing red blood cells with ammonium chloride-potassium(ACK) lysis buffer (0.15M NH4Cl, 10 mM KHCO₃, 0.1 mM Na₂EDTA, pH 7.2). Tcells were enriched by using the PAN T cell isolation kit (MiltenyiBiotec, Auburn, Calif.) and by depletion of magnetically labeled cellsemploying AutoMACS (Miltenyi Biotec). Twelve hours post-MPTPintoxication, both B10.BR and B6 mice received intravenous (i.v.)injections of 5×10⁷ spleen cells (SPC) in 0.25 ml of Hanks' balancedsalt solution (HBSS). B10.BR mice also received 2.5×10⁷ purified Tcells. SCID mice were reconstituted with i.v. injections of 8×10⁷unfractionated SPC populations from WT B6 mice. RCS-SCID mice wererested for 4 wks prior to MPTP intoxication.

³H-Thymidine in Vitro Proliferation Assays

Samples of pooled immunized donor cells used for adoptive transfer weretested for their proliferative capacity by ³H-thymidine incorporationafter stimulation with either immunizing or irrelevant antigen. DonorSPC were plated at a density of 2×10⁶ cells/ml complete RPMI tissueculture media [RPMI 1640 supplemented with 10% fetal bovine serum (FBS),2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 1× nonessential aa,55 μM 2-mercaptoethanol, 100 U/ml penicillin, and 100 μg/ml streptomycin(Mediatech Inc., Herndon, Va.)]. SPC from PBS, 4YSyn, and N-4YSynimmunized mice were stimulated with 0, 1, 10, 50 μg/ml of immunizingantigen, 4YSyn or N-4YSyn, and cultured at 37° C. for 5 days. Cells werepulsed with 1 mCi ³H-thymidine/well for the final 18 hours of culture,harvested onto glass fiber plates, and counted by β-scintillationspectrometry (TopCount, Packard-PerkinElmer Instruments, Wellesley,Mass.).

Western Blot Analysis

Ventral midbrain (VMB) and lymphoid organ protein extracts (80 μg/lane)were separated by 16% SDS-PAGE (Invitrogen) and transferred for 45minutes onto 0.2 mm PVDF membranes (Millipore, Bedford, Mass.).Membranes were probed with rabbit antibodies to NT (1:2000; Chemicon) ormonoclonal rat antibodies to myelin basic protein (MBP, 1:1000,Chemicon) or guinea pig antibodies to α-Syn (1:1000; Ab-1, Oncogene/EMDBiosciences). Appropriate HRP-conjugated secondary antibodies (SantaCruz Biotechnology, Santa Cruz, Calif.) were used to visualize blotsusing SuperSignal® West Pico Chemiluminescent substrate and CCL-XPosurefilm (Pierce Biotechnology, Inc., Rockford, Ill.). Immunoblots werestripped and reprobed with antibodies to α-actin (1:5000; Chemicon,) asan internal control.

Identification of α-Syn in MPTP-CLN

Anti-N-α/β-syn (clone nSyn12, mouse ascites, Upstate) that specificallyrecognizes N-α-Syn (14.5 kD) and N-β-Syn (17 kD) but not non-nitratedα/β-Syn was used for immunoprecipitation (IP). VMB and CLN from PBS orMPTP treated mice were dissected out, homogenized in ice-cold RIPAbuffer, pH 7.4 and centrifuged at 10,000×g for 10 minutes at 4° C. toremove cellular debris. Protein A/G PLUS-Agarose beads (Santa CruzBiotechnology) were added to 2 mg total cellular protein, incubated for1 hour at 4° C. Beads were centrifuged at 1,000×g for 5 minutes at 4° C.The supernatant was incubated with 40 ml anti-N-α-Syn overnight at 4° C.on a rotating device, and then with Protein A/G PLUS Agarose beads for 1hour on a rotating device at 4° C. Immunoprecipitates were collectedafter centrifugation at 1,000×g for 5 minutes at 4° C., washed once withRIPA buffer and twice with PBS, resuspended in 40 ml of 1×electrophoresis sample buffer.

N-α-Syn IP samples were fractionated by large format 16% TricineSDS-PAGE (Jule Inc., Milford, Conn.; BIORAD Laboratories, Inc, LosAngeles, Calif.) at constant voltage for 8-10 hours. The gel was stainedwith highly sensitive SYPRO Ruby stain (Invitrogen) and scanned atexcitation (400 nm) and emission (630 nm) wavelengths using Typhoonscanner (Amersham Biosciences, Piscataway, N.J.) to visualize theprotein bands. Small gel fragments (3-4 mm) corresponding to molecularweight (12-18 kD) were excised from each lane of the same gel stainedwith Coomassie. In brief, gel pieces were destained for 1 hour at roomtemperature using 100 ml of 50% ACN/50 mM NH₄CO₃. Gel pieces were driedand incubated with trypsin in 10 mM NH₄CO₃ (Promega, Madison, Wis.)overnight at 37° C. Peptides were extracted by washing gel pieces twicewith 0.1% TFA and 60% ACN. Dried samples were resuspended in 12 ml of0.1% formic acid in water for automated injection. All samples werepurified using ZipTip® (Millipore) prior to MS analysis. In-gel trypsindigested proteins were fractionated on microcapillary RP-C18 (Ciborowskiet al. (2004) J. Neuroimmunol., 157:11-16). The resulting peptides weresequenced using Electrospray Ionization (ESI)-LC MS/MS (Proteome XSystem with LCQDecaPlus mass spectrometer, thermoElectron, Inc., SanJose, Calif.) with a nanospray configuration. The spectra obtained fromLC-MS/MS analysis were searched against the NCBI.fasta rodent proteindatabase using SEQUEST search engine (BioWorks 3.2 SR software fromThermoElectron, Inc, San Jose, Calif.). Criteria for high confidenceprotein identification were used as previously published (Ciborowski etal. (2004) J. Neuroimmunol., 157:11-16; Enose et al. (2005) Glia51:161-172; Ricardo-Dukelow et al. (2007) J. Neuroimmunol., 185:37-46;Ciborowski et al. (2007) Virology 363:198-209; Glanzer et al. (2007) J.Neurochem., 102:627-45; Kadiu et al. (2007) J. Immunol., 178:6404-6415).

Flow Cytometry

Single cell suspensions were prepared from deep CLN from C57BL/6 mice20-24 hours post PBS or MPTP (18 mg/kg) intoxication. Cell suspensionswere analyzed for cell surface expression of CD11c, CD11b, and MHC classII (I-A^(b)). Also, prior to adoptive transfers, cell populations fromimmunized donors were stained for T cells using PE conjugated anti-mouseCD3ε (BD Pharmingen) and B cells with FITC conjugated anti-mouse CD19(BD Pharmingen). Analysis was performed with a FACSCalibur™ flowcytometer interfaced with CellQuest™ software (BD-Biosciences,Immunocytometry Systems, San Jose, Calif.).

Determination of N-4YSyn-mediated Toxicity In Vitro

For proliferation analyses, purified T cells from naive B6 mice wereplated with SPC irradiated at 3000 rad (1:3) at 2×10⁶ cells/ml incomplete RPMI tissue culture media and activated with anti-CD3 (0.5μg/ml, 145-2C11; BD Pharmingen) in U-bottom 96-well tissue cultureplates. Graded concentrations of 4YSyn or N-4YSyn were added toquadruplicate wells. After activation for 3 days, ³H-thymidineincorporation was performed as described previously.

To assess α-Syn-mediated cytotoxicity, purified T cells were stimulatedwith anti-CD3 and cultured at a density of 1×10⁶ cells/ml for 24 hoursin media alone or in the presence of 4YSyn or N-4YSyn at concentrationsof 1, 3, 10, or 30 μg/ml. Cells were stained with PI, washed and thepercentages of PI⁺ dead cells and MFI were analyzed by flow cytometry.

Macrophage and MES 23.5 Cultures

BMM were prepared from C57BL/6 adult male (6-12 weeks old) mice. Theanimals were sacrificed by CO2 asphyxiation. Single cell suspensions ofbone marrow cells were obtained from femur bone marrow cavities afterflushing with HBSS, and red blood cells lysed with ACK buffer. The bonemarrow cells were cultured in complete DMEM medium (Dulbecco's ModifiedEagles Media supplemented with 10% FBS, 2 mM L-glutamine, and 1%penicillin/streptomycin) containing 2 μg/ml macrophage colonystimulating factor (MCSF), a generous gift from Wyeth Pharmaceuticals(Cambridge, Mass.) in a 5% CO₂/37° C. incubator. Nonadherent cells wereremoved from flasks at 1, 4, and 7 days by successive DMEM washes.Adherent BMM were harvested and replated for experiments following 7-14days of culture. Cells from the MES 23.5 dopaminergic cell line werecultured in 75-cm² flasks in DMEM/F12 with 15 mM HEPES (Invitrogen)containing N2 supplement (Invitrogen), 100 U/ml of penicillin, 100 μg/mlstreptomycin, and 5% FBS. Cells were grown to 80% confluence thenco-cultured with BMM in serum free MEM/F12 at a density of 1×10⁵ cells(1:1) on sterile glass coverslips.

N-α-Syn SPC-Induced Microglia Cytotoxicity

SPC isolated from N-4YSyn (10 μg) immunized B6 mice were cultured inRPMI media and activated in vitro for 4 days with N-4YSyn (1 μg/ml). MES23.5 cells or macrophages alone or MES 23.5 and macrophage co-cultureswere stimulated with aggregated N-α-Syn (1.45 μg/ml) alone and incombination with either activated SPC or supernatants obtained fromactivated SPC for 24 hours. Unstimulated cultures served as controls.Assays for viable and dead cells were performed with Live/DeadViability/Cytotoxicity kit (Invitrogen) according to the manufacturer'sprotocol and viewed under a fluorescence microscope (Nikon Eclipse E800,Buffalo Grove, Ill.). Images were captured at 100× magnification andquantification of live (green) and dead (red) counts was performed from4-8 different fields.

Cytokine Array

Triplicate co-cultures of antigen presenting cells (APC) and T cellsfrom PBS, 4YSyn- and N-4YSyn-immunized mice were stimulated with 4YSynor N-4YSyn. After 24 hours of culture, 50 μl samples were collected,centrifuged, and supernatants frozen at −80° C. until utilized. Frozensupernatants were thawed only once and analyzed using the BD CytometricBead Array Mouse Th1/Th2 Kit (BD Biosciences, San Jose, Calif.)according to the manufacturer's instructions.

Statistical Analysis

All values are expressed as mean±SEM. Differences among normallydistributed means were evaluated by Student's t test for two groupcomparisons or one-way ANOVA followed by Bonferroni post-hoc tests forpairwise comparisons amongst multiple data sets (Statistica v7,Statsoft, Tulsa, Okla., and SPSS v13, SPSS, Inc., Chicago, Ill.) andwere considered significant at p<0.05 unless otherwise indicated.Kolmogorov-Smimov (K-S) analysis was performed for flow cytometric dataanalysis.

Results CNS Antigens Drain to CLN Following MPTP Intoxication

To determine if CNS antigens drain to CLN during establishedneurodegeneration of the nigrostriatal pathway, their presence inventral midbrain (VMB), cervical, axillary, inguinal, mesenteric lymphnodes and spleens were determined 24 hours after MPTP-intoxication inC57BL6 mice. The presence of unmodified α-Syn was demonstrated in VMBand CLN (FIG. 1A) as well as other lymph nodes and in the spleen fromphosphate-buffered (PBS)— or MPTP-treated mice in anti-α-Syn-probedimmunoblots. These findings also confirmed the expression of unmodifiedα-Syn amongst cells of hematopoietic lineage (Shin et al. (2000) Mol.Cells, 10:65-70). N-α-Syn IP showed that N-α-Syn was present in the CLNof MPTP-treated, but not PBS-treated mice as similar molecular weightbands were observed from gels probed with SYPRO® Red and Western blotsperformed with α-Syn antibodies (FIG. 1B). To validate the presence ofNT-modified α-Syn after MPTP treatment, N-α-Syn immunoprecipitates wereobtained after in-gel tryptic digestion of 12-18 kD fragments acquiredfrom the VMB and CLN and sequenced by LC-MS/MS. This regions was chosenas it represents the molecular mass ranges of oxidized α-Syn (Weinreb etal. (1996) Biochemistry 35:13709-3715; Hodara et al. (2004) J. Biol.Chem., 279:47746-47753; Dufty et al. (2007) Am. J. Pathol.,170:1725-1738; Hasegawa et al. (2002) J. Biol. Chem., 277: 49071-49076;E1-Agnaf et al. (2003) Faseb J., 17: 1945-194). Sequence analysisdemonstrated α-Syn peptides (yellow highlighted sequences, FIG. 1C) inthe VMB from both PBS- and MPTP-treated mice but exclusively in the CLNof MPTP-intoxicated mice (Table 1). α-Syn peptides were identifiedat >99.999% confidence (Table 1). Western blot analysis of lymphoidtissue homogenates using rabbit NT antibodies detected a single bandwith a molecular mass of ˜16-18 kD, which is comparable to that ofα-Syn, in CLN from MPTP-intoxicated animals, but not in other lymphnodes or spleen (FIG. 1D). These results were confirmatory for thepresence of N-α-Syn in the draining CLN. Another CNS antigen, MBP wasalso detected only in the CLN of MPTP intoxicated animals (FIG. 1D).NT-modified proteins and MBP were absent in lymph nodes and spleens ofcontrol (PBS-injected) mice. These data suggest that brain proteinsreleased as a consequence of nigrostriatal injury, drain to the deepCLN, placing them in organs associated with efficient presentation ofantigen. To demonstrate the functional significance of theseobservations, single cell suspensions were prepared from CLN isolatedfrom MPTP animals and controls, and analyzed by flow cytometry for MHCclass II expression on CD11b⁺ APC (FIG. 1E). Increased frequencies ofCD11b+/MHC class II⁺ in MPTP-treated mice compared to PBS controls wastaken as evidence of leukocyte activation in the deep CLN followingMPTP-induced nigrostriatal injury. Supporting the induction of a α-Synspecific immune response, sera from WT B6 mice 21 days after chronicMPTP intoxication were analyzed for anti-α-Syn IgG and compared toanimals that received PBS. Serum levels of α-Syn antibodies in miceexposed to MPTP were significantly increased (FIG. 1F). Together, theseresults demonstrate that NT-modified α-Syn draining into the deep CLN iscapable of eliciting a peripheral immune response.

TABLE 1 Probabilities (p values) of protein sequence matches within12-18 kD bands from anti-N-α/β-synuclein immunoprecipitation and LCMS-MS analyses of VMB and CLN from PBS- or MPTP-treated mice. P valuefor protein matches from VMB MPTP Protein Match PBS MPTP PBS MPTPα-synuclein 5.9 × 10⁻⁷ 7.7 × 10⁻⁶ 1.0 × 10⁻⁶ β-synuclein 4.3 × 10⁻⁷ 3.8× 10⁻⁹ myelin basic protein 3.4 × 10⁻⁵ MHC class I antigen 4.0 × 10⁻³immunoglobulin heavy chain variable 5.8 × 10⁻⁵ region chemokine-likefactor super family 3.8 × 10⁻⁴ five variant 4 ribosomal protein S14 5.6× 10⁻⁶ Tesp4 protein 1.8 × 10⁻⁵ A chain A, complex of the second 2.0 ×10⁻⁴ 8.7 × 10⁻⁵ kunitz domain of tissue factor pathway inhibitorstructural constituent of ribosome 6.3 × 10⁻⁵ parotid secretory protein2.6 × 10⁻⁴ ribosomal protein S14 4.9 × 10⁻⁴ Similar to NADHdehydrogenase 5.9 × 10⁻⁴ (ubiquinone) 1 beta subcomple mediator of RNApolymerase II 9.9 × 10⁻⁴ transcription, subunit 8 homolog isoformcAMP-dependent protein kinase, 1.3 × 10⁻³ alpha-catalytic subunit (PKAC-alpha) step II splicing factor SLU7 2.4 × 10⁻³ parotid secretoryprotein 3.5 × 10⁻³ Similar to protease, serine, 3 1.2 × 10⁻⁵hypothetical protein LOC320696 4.5 × 10⁻³ Unknown (protein for MGC:116262) 2.9 × 10⁻⁶ unnamed protein product (16288 kD) 1.6 × 10⁻⁴ unnamedprotein product (25311 kD) 1.1 × 10⁻⁵ unnamed protein product (27163 kD)2.4 × 10⁻⁴ unnamed protein product (58621 kD) 1.2 × 10⁻⁵ 8.2 × 10⁻⁵unnamed protein product (65626 kD) 2.7 × 10⁻⁶ 1.4 × 10⁻⁵ 2.5 × 10⁻⁵pancreatic trypsin 1 3.7 × 10⁻⁵ 2.7 × 10⁻⁶ 1.5 × 10⁻⁶ 1.3 × 10⁻⁴ trypsin10 2.9 × 10⁻³ 1.8 × 10⁻⁴ 5.1 × 10⁻⁵ 8.6 × 10⁻⁵ trypsinogen 7 2.2 × 10⁻⁵2.6 × 10⁻⁶

Adaptive Immunity Participates in MPTP-Nigral Degeneration

The presence of NT modifications of α-Syn in draining lymphatic tissuefollowing MPTP-induced nigrostriatal injury, along with evidence oflymphoid-associated APC activation provided support for antigenpresentation to T cells and subsequent immune responsiveness. Tosubstantiate this, it was explored whether an endogenous adaptive immunesystem was required for MPTP-induced nigrostriatal degeneration. B6 WTmice, B6 SCID mice, and B6 SCID mice reconstituted with 10⁸ B6 WTsplenocytes (SPC) (RCS-SCID) were treated with PBS or a chronic MPTPregimen. Mice were sacrificed at 21 days after the last MPTP injection,and VMB sections immunostained for tyrosine hydroxylase (TH), therate-limiting enzyme in dopamine synthesis (FIG. 2A, left panels). Thenumbers of TH⁺ neurons in the SN showed a 33% reduction in WT B6 animalsthat received MPTP compared to those that received PBS (FIG. 2B). Nosignificant difference in the numbers of TH⁺ neurons was observed inMPTP-treated SCID mice compared to SCID mice that received PBS (FIG.2B). In contrast, immune reconstituted SCID mice (RCS-SCID) treated withMPTP showed significantly fewer TH⁺ neurons compared to the SCID MPTPgroup (FIGS. 2A and 2B). To validate the reconstitution of RCS-SCIDmice, spleens were immunostained for CD3⁺ T cell distribution (FIG. 2A,right panels). T cell repopulation was confirmed by the presence of CD3⁺T cells in the periarteriolar lymphoid sheath of RCS-SCID mouse spleens(FIG. 2A), VMB and cerebellum control sections of WT, SCID, and RCS-SCIDmice treated with MPTP were immunostained for T cells using antibodiesagainst CD3, CD4, and CD8. CD3 immunostaining of MPTP-treated B6 micedemonstrated CD3⁺ cells in the VMB beginning at day 0, present at day 4after MPTP intoxication (FIG. 2C) that persisted to day 14. Both CD4⁺and CD8⁺ subpopulations were also present in VMB of only MPTP-treatedanimals at 4 and 14 days (FIG. 2C). No T cell accumulation was observedin PBS or MPTP-treated SCID mice at any time point, whereas CD3⁺ T cellaccumulations in VMB of RCS-SCID mice after MPTP-treatment wereidentified. Cerebellar tissue of MPTP animals had ≦1 CD3⁺ T cell perhigh power field examined present suggesting specific cell entry intoaffected regions. Taken together, these data support the occurrence ofan adaptive immune response triggered by modified CNS antigens thatmodulates the vulnerability of the dopaminergic neurons to MPTP throughthe migration of T cells into the CNS.

Prediction of Mouse N-α-Syn Specific T Cell Epitopes

To test the probability of N-α-Syn induced adaptive immune responses,the numbers of predicted α-Syn specific T cell epitopes were comparedwith the propensity to bind class I MHC grooves (Peters et al. (2006)PLoS Comput. Biol., 2:e65) for the murine MHC haplotypes, H-2^(k) andH-2^(b) (see Table 2). However, since the last 40 aa of the mouse α-Syncontains 4 Tyr residues available for nitration, the analysis focused onthis C-terminal α-Syn fragment. Table 2 shows that H-2K^(k) epitopeshave a superior ability to bind with high and intermediate affinityα-Syn-derived 8-11-meric peptide fragments derived from the whole α-Synmolecule or from its C-terminal 38-meric fragment. These datademonstrate significant T cell induction potential. In fact, α-Syn has100 potential T cell epitopes, 73 of which contain Tyr that werepredicted to bind H-2K^(k) molecules, while only 8 and 15 potentialepitopes may bind H-2 D^(b) and H-2 K^(b) molecules, respectively. Thiswas also for nitrated epitopes containing Tyr residue including thosewith a Tyr residue within the central region of the epitope thatpresents prominently to the T cell receptor. These epitopes do notcontain anchor aa that mediate binding to the MHC groove. Dramaticdifference in the number of Tyr-containing T cell epitopes fromC-terminal segment of α-Syn predicted to bind H-2K^(k) but not any ofH-2^(b) molecules (Table 2, data in brackets) suggests a potentialpreference for H-2K^(k) versus H-2^(b) mice to induce T cell responsesto nitrated C-terminal fragments of α-Syn. These fragments, if facing Tcell receptors, have greater chances of inducing MHC class I-restrictedCD8+ T cells due to lack of negative selection against N-α-Syn epitopesin the embryonic thymus. Further epitope prediction analysis revealed anumber of 15-meric epitopes, including Tyr-containing peptides fromC-terminal, that can bind with increased affinity class II MHC groove,thus increasing the propensity of inducing MHC class II-restricted CD4⁺T cells specific for Tyr-containing α-Syn C-terminal fragments.Therefore, mice expressing MHC class I and II molecules of H-2khaplotypes are capable of generating immune responses to NT-modifiedα-Syn. Interestingly, that α-Syn C-terminal fragment contains severalTyr-containing peptides with predicted significant binding affinity forIA^(k) and IA^(b) MHC molecules (18 and 14 epitopes, respectively)suggests a significant potential for CD4⁺ T cells of mice expressingIA^(k) or IA^(b) to respond to nitrated epitopes from α-Syn C-terminal.

TABLE 2 Numbers of putative α-Syn epitopes for presentation to T cellreceptors predicted from the binding potential of MHC class I and IImolecules for the aa sequence of α-Syn. Number of predicted T-cellepitopes from the 38-mer C-terminal fragment of α-Syn are in brackets.Predicted Number of predicted epitopes for: binding of ^(a)MHC class I^(b)MHC class II epitopes K^(k) D^(b) K^(b) IA^(k) IE^(k) IA^(b) All 143(100) 72 (1)   73 (13) 116 (32)  59 (8)  129 (32)  High 29 (26) 1 (0)  1(1) 19 (12) 0 (0) 2 (2) Intermediate 71 (58) 7 (0) 14 (6) 55 (13) 6 (1)70 (16) Low 43 (16) 64 (1)  58 (6) 42 (7)  53 (8)  57 (14) ContainingTyr 101 (77)  50 (1)  48 (8) 34 (23) 8 (5) 33 (24) High 24 (21) 1 (0)  1(1)  9 (10) 0 (0) 2 (2) Intermediate 49 (43) 7 (0) 12 (4) 19 (8)  0 (0)16 (12) Low 28 (13) 42 (1)  35 (3) 6 (5) 8 (5) 15 (10) ^(a)For class IMHC binders three following grades for scoring included: low immunogenicepitopes, scores <−3; mild immunogenic epitopes, scores >−3 but <−2;high immunogenic, scores >−2; all computations were done using theImmune Epitope Database and Analysis Resource (IEDB) (immuneepitope.org)and integrative epitope prediction tool [proteasomes cleavage,Transporter associated with Antigen Processing (TAP) binding, processingand MHC binding]. ^(b)For class II MHC binders, scoring grades werebased on predicted IC₅₀ values and were: low (1000 nM-5000 nM),intermediate (200-1000 nM) and high (<200 nM) for groove bindingprediction by MHCPred. This prediction algorithm considers peptides withpredicted IC₅₀ > 5000 nM as non-binders (Guan et al. (2003) Appl.Bioinformatics 2: 63-66; Guan et al. (2003) Nucleic Acids Res., 31:3621-3624).

Purification and Nitration of Recombinant α-Syn

Based on the above findings, it was hypothesized that in PD, NTmodifications of a-Syn could be a key step converting the endogenousprotein to an immunogen. Here, the C-terminal 40 aa α-Syn fragment(4YSyn) was used as it contains all four tyrosine residues that arenitrated, thus limiting the possible specificities of epitopes capableof generating an immune response. For this, the mouse cDNA encoding thefinal 40 aa was cloned into the bacterial pET-28a His-tag expressionvector and recombinant protein expressed in BL21 E. coli followingisopropyl-β-D-thiogalactopyranoside (IPTG) induction. Expression of therecombinant protein exhibited no apparent toxicity to the bacterialexpression system. Affinity-purified 4YSyn peptide from E. coli lysateswas detected as a prominent single band using silver staining on 12%polyacrylamide gel (FIG. 3B) and by Western blot using a polyclonalantibody raised against aa 120-140 of α-Syn (FIG. 3C). Reverse-phasehigh performance liquid chromatography (RP-HPLC) analysis of isolated4YSyn products demonstrated purities equal to or in excess of 97%. NTmodifications of 4YSyn peptide (N-4YSyn) after peroxynitrite nitrationwas confirmed by Western blot using mouse monoclonal anti-NT antibody(FIG. 3C).

Homogeneity of purified 4YSyn and its modified forms (aggregated andnitrated) was assessed based on: 1D SDS-PAGE (FIG. 3B), Western blot(FIG. 3C), and matrix-assisted laser desorption ionization-time offlight (MALDI-TOF) mass spectrometry (FIG. 3D). The predominant peak for4YSyn by MALDI-TOF analysis was 6592 m/z, which corresponded to the 6718expected mass of purified recombinant α-Syn within <2% mass accuracy(FIG. 3D and Table 3). To provide proof that the mass discrepancyoriginated from recombination errors within the His-tag region obtainedduring protein purification, but not within the biologically activeportion of the molecule, the recombinant 4YSyn protein was digested withtrypsin and measured masses of resulting fragments using MALDI-TOF. Theobserved masses of the generated fragments were Arg-cleaved 4YSyn andLys-cleaved 4YSyn. These corresponded to the expected masses with 0.07%of mass accuracy. Next, the mass of native 4YSyn was compared toN-4YSyn. The oxidized peptide or its trypsin cleaved fragments revealeda 184 D mass increase that is analogous to the expected mass of 4 nitrogroups corresponding to 4 NT-residues (FIGS. 3A and 3D). Based on theseobservations, it was concluded that reaction of 4YSyn withperoxynitrite, under the conditions used in this study, efficientlynitrated all four available Tyr residues in 4YSyn.

TABLE 3 Theoretical and observed masses of 4YSyn, N-4YSyn and trypticdigest fragments. Peptide Theoretical Mass (D) Observed Mass (D)His-Tagged 4YSyn 6718 6592 His-Tagged N-4YSyn 6902 6775 Arg Cleaved4YSyn 4836 4833 Lys Cleaved 4YSyn (K) 4238 4241 Arg Cleaved N-4YSyn 50205073 Lys Cleaved N-4YSyn 4422

N-4YSyn Induces Specific Immune Responses in B10.BR Mice

To test the predictions of immune responses to N-α-Syn, B10.BR (H-2^(k))mice were immunized with N-4YSyn, 4YSyn, or PBS each emulsified incomplete Freund's adjuvant (CFA) (FIG. 4A). Fourteen days following theinitial immunization, mice were boosted with their respective immunogensemulsified in incomplete Freund's adjuvant (IFA). Five days later, micewere sacrificed and SPC were tested for antigen-specific T cellproliferative responses to N-4YSyn or 4YSyn. Stimulation with 4YSynyielded no significant immune responses regardless of whether mice wereimmunized with adjuvant containing PBS, 4YSyn or N-4YSyn (FIG. 4B). Incontrast, significant proliferative responses were afforded from SPC ofmice immunized with N-4YSyn and challenged in vitro with N-4YSyn, butnot 4YSyn. Moreover, N-4YSyn stimulated SPC from mice immunized withadjuvant containing 4YSyn or PBS failed to induce significantproliferative responses. These data indicate that immunization withN-4YSyn, but not 4YSyn is capable of inducing antigen specific immuneresponses to NT-modified CNS antigens.

Adoptive Transfer of N-4YSyn SPC and T Cells Exacerbates MPTP-InducedMicroglial Activation and Dopaminergic Neuronal Death

In light of the fact that modified α-Syn is capable of evading toleranceand inducing reactive T cells, it was tested whether modifiedα-Syn-activated T cells could exacerbate MPTP-induced dopaminergicneurodegeneration. The experimental scheme for adoptive transfer of SPCor purified T cells from immunized animals is outlined in FIG. 4A. Forthese studies, B10.BR (H-2^(k)) donor mice were immunized and boostedwith N-4YSyn or 4YSyn, and SPC were adoptively transferred toMPTP-treated syngeneic recipients. To delineate effects due specificallyto T cells, CD3+ T cells were enriched by negative selection andtransferred to an additional group of MPTP-treated animals. Flowcytometric analysis showed that the enriched population from N-4YSynmice was 94% CD3+ T cells (FIG. 5A). Adoptive transfer of purified Tcells from N-4YSyn immunized donors to MPTP intoxicated mice revealedCD3⁺ T cell infiltrates in the SNpc on day 2 after MPTP treatment (FIG.5B).

MPTP treated mice showed fluorescent neurons within the SN using thedegenerating cell marker Fluoro-Jade C by day 2, but not by day 7 (FIG.6, left and middle panels) confirming previous kinetic data regardingMPTP-induced nigral neuronal death obtained by silver stainingtechniques (Jackson-Lewis et al. (1995) Neurodegeneration 4:257-269).MPTP treated mice that received immune cells from N-4YSyn immune miceshowed more Fluoro-Jade C stained neurons within the SN by day 2 thanMPTP-intoxication alone, and, in contrast to the latter, Fluoro-Jade Cstained neurons within the SN by day 7 as well. In the PBS controlgroup, no Fluoro-Jade C stained neurons were observed at any time point.Following MPTP administration, microgliosis is striking and immediate.The initial time course studies are in line with these findings and showthat the microgliosis and dopaminergic neurodegeneration in B10.BR miceare virtually resolved respectively by days 4 and 7 post-MPTP injection(FIG. 6, right and middle panels, respectively). However, adoptivetransfer of SPC from B10.BR mice, regardless of immunization protocol,was associated with a persistent microglial response, as evidenced byquantitative morphology with Mac-1 immunostaining (FIG. 6, right panel).Counts of Mac-1+ microglia were greatest (p<0.0001) in MPTP mice treatedwith SPC from N-4YSyn immunized mice [84.1±7.0/mm² (mean±SEM)] comparedto those from mice treated with MPTP and SPC from 4YSyn immunized mice(26.9±3.5/mm²), MPTP alone (27.7±3.2/mm²), or PBS (0.7±0.3/mm²). Thesedata indicate that the adaptive immune components of H-2^(k) micefollowing MPTP administration contribute to the neuroinflammatoryphenotype seen in these animals.

Based on these findings, it was investigated whether the immune responsemediated by N-α-Syn affects degeneration of dopaminergic cell bodies inthe SNpc. To test this, MPTP-intoxicated B10.BR mice received 5×10⁷donor SPC from mice immunized with PBS, 4YSyn, or N-4YSyn, or 2.5×10⁷enriched T cells from 4YSyn-immunized donors. PBS- and MPTP-treated micethat did not receive cells served as controls for no neuronal loss andloss attributable to MPTP treatment alone, respectively. PBS-treatedmice that received N-4YSyn immunized donor SPC served as additionalcontrols. VMB sections were obtained from mice at 2, 7, and 28 daysfollowing MPTP treatment and immunostained for TH (FIG. 7A).Stereological analysis showed that MPTP induced a 45% reduction of SNTH⁺ neurons compared to PBS controls (FIG. 7B). Similar results wereobserved in MPTP-injected mice that received SPC from PBS or 4YSynimmune donors (MPTP/PBS/SPC and MPTP/4YSyn/SPC, respectively). Incontrast, recipients that received immune N-4YSyn SPC (MPTP/N-4YSyn/SPC)or N-4YSyn T cells (MPTP/N-4YSyn/T Cells) exhibited significantlygreater reductions of SNpc TH⁺ neurons (64 and 63%, respectively)compared to all other MPTP-treated animals (FIG. 7B). PBS-treated micethat received immune SPC from N-4YSyn immunized donors showed no changein TH⁺ neuron numbers, demonstrating the necessity for an initiatingneuronal insult. Significant effects from any treatment were notobserved among the numbers of non-dopaminergic neurons (Niss1+TH−).Correlation analysis of total Niss1+ neurons compared to TH+ and TH−neurons demonstrated that the number of total neurons stronglycorrelated with numbers of TH+ neurons (r=0.981, p<0.0001) compared tonumbers of TH− neurons (r=0.522, p=0.004). This confirmed thatdifferences in TH+ neuron counts are due to differences in numbers ofstructurally intact neurons and eliminates the possibility thatdifferences resulted from the down-regulation of TH itself. Thus, thesedata demonstrate that adaptive immune responses against the nitratedform of α-Syn exacerbated MPTP-induced nigrostriatal degeneration.

Based on MHC binding affinity algorithms, mice expressing H-2^(b) werepredicted to respond poorly to α-Syn epitopes; yet 21 days after chronicMPTP-intoxication, B6 mice that express the H-2^(b) haplotype yieldedsignificant antibody responses to N-α-Syn. This suggested that miceexpressing H-2^(b) have the potential to develop immune responses toN-α-Syn that may affect disease progression. To assess that possibility,B6 (H-2b) mice were immunized and boosted with 4YSyn or N-4YSyn eitherin PBS or emulsified in adjuvant (FIG. 8A). Five days after the finalboost SPC were harvested, assessed for antigen specific lymphocyteproliferation, and adoptively transferred to MPTP-intoxicatedrecipients. Stimulation of SPC from PBS-treated controls indicated thatneither 4YSyn nor N-4YSyn induced significant proliferation above mediumbackground levels (FIG. 8B). In contrast, stimulation of SPC fromN-4YSyn/PBS immunized mice with N-4YSyn induced a significant lymphocyteproliferative response indicating that immunization with N-4YSyn/PBS inthe absence of adjuvant is capable of inducing an antigen specificadaptive immunity.

TH stained sections of VMB from MPTP-intoxicated mice that received SPCfrom donors immunized with N-4YSyn either in PBS excipient or adjuvantshowed significant dopaminergic neuronal losses compared to MPTP-treatedmice or those that received SPC from 4YSyn immunized mice (FIG. 9A),suggesting 4YSyn immunization increased the MPTP-induced lesion. Next,the sections were assessed by stereological analysis to obtain estimatesof dopaminergic neuronal survival and loss after treatment compared toPBS-treated mice. MPTP-intoxication of B6 mice induced 43% loss of TH+nigral neurons compared to PBS-treated controls (FIG. 9B). Numbers ofdopaminergic neurons from MPTP mice treated with SPC from 4YSynimmunized mice were not significantly different compared to MPTP-treatedmice and showed a similar 42% loss of neurons. However, adoptivetransfer of SPC from N-4YSyn/Adjuvant immune donors significantlyincreased MPTP-induced dopaminergic neuron loss to 58%. Interestingly,SPC from mice immunized with N-4YSyn without adjuvant induced a 69% lossof nigral TH+ neurons after MPTP intoxication, which was significantlygreater than losses due to SPC from 4YSyn- or N-4YSyn/Adjuvant immunizeddonors. Neither MPTP treatment nor adoptive transfer of immune SPCsignificantly affected numbers of non-dopaminergic neurons. Thus, takentogether these data demonstrate that N-α-Syn, but not unmodified α-Syn,has the capacity to induce specific immune responses by whichexacerbates neuronal loss in the context of dopaminergicneurodegeneration. Moreover, these results in H-2^(b) mice, predicted toprovide poor immune response, suggests that epitopes modified byinflammatory processes may function unlike their tolerated unmodifiedself-analogues to induce immune responses to levels sufficient to alterdisease progression.

Nitrated α-Syn Inhibits Proliferation of Anti-CD3 Activated T Cells

It was observed that antigen specific proliferative responses wereinhibited by N-4YSyn in a dose dependent manner (r²=0.96, p=0.002).Significant inhibition was not seen by 4YSyn (r²=0.53, p=0.273). To ruleout an antigen specific suppressive effect, purified T cells obtainedfrom naive mice were stimulated with anti-CD3 for 24 hours in media orin the presence of 4YSyn or N-4YSyn at concentrations of 1, 3, 10 and 30μg/ml. N-4YSyn inhibited proliferation of anti-CD3 stimulated T cells ina dose dependent fashion (r²=0.6803, p<0.0001). A significant inhibitionof 31 and 47% was observed when stimulated T cells were co-cultured atN-4YSyn concentrations of 10 and 30 mg/ml (FIG. 10). In contrast,proliferation of anti-CD3 stimulated T cells was not inhibited by 4YSyn(p=0.435) or by increasing 4YSyn concentration (r²=0.1554, p=0.0854).Moreover, the inverse effect on anti-CD3 activated T cells withincreasing N-4YSyn concentration was greater (p=0.016) compared to thatinduced by 4YSyn. Second, to assess whether this effect was due to acytotoxic mechanism, anti-CD3 stimulated T cells were stained for themembrane impermeant DNA dye, propidium iodide (PI) that is excluded fromintact, living cells, and analyzed by flow cytometry. T cells culturedin the presence of N-4YSyn exhibited a dose-dependent increase in themean fluorescent intensity (MFI) of PI (r²=0.9261, p=0.0008) and in thepercentage of PI⁺ dead T cells (r²=0.9743, p=0.0018) (Table 4). Incomparison, percentages of PI stained dead T cells cultured with 4YSynand the PI MFI were not different from the basal level in absence ofeither of the peptides and did not change with increasing 4YSynconcentration (r²=0.5983, p=0.1249, and r²=0.5016, p=0.1807,respectively). Taken together, increasing N-4YSyn concentrationsstrongly correlated with inhibition of T cell proliferation, percent PI⁺T cells, and increased MFI for PI (r>0.945, p<0.0154 for all comparisonscombined). In contrast, no significant correlations were associated withincreasing 4YSyn concentrations. Thus, cytotoxic interference of immuneresponse induction by N-α-Syn may affect disease progression as theseprocesses are shared by both effector and regulatory T cells.

TABLE 4 N-4YSyn-induced cytotoxicity of stimulated T cells. ^(a)T cellConc. ^(e)Index of Treatment μg/ml ^(b)% PI⁺ T cells ^(c)MFI ^(d)DSimilarity Medium 5.7 33.0 * 0 4YSyn 1 4.9 32.3 0.08 3.8 3 6 34.1 0.084.2 10 4.7 25.2 0.1 5.1 30 4.2 26.8 0.16 8.1 N-4YSyn 1 15.9 42.8 0.3115.7 3 17.9 58.7 0.33 15.6 10 47.8 71.6 0.64 30.9 30 91.3 106.1 0.8743.4 ^(a)Anti-CD3 stimulated T cells were cultured for 24 hours in mediaalone or in the presence of different concentrations of 4YSyn orN-4YSyn, stained with 2 μg/ml of the vital dye, PI, washed, and assessedby flow cytometric analysis for uptake of PI. ^(b)Percentage of T cellssusceptible to PI permeation as determined by flow cytometric analysis.^(c)Mean fluorescence intensity of PI-stained T cells. ^(d)D statisticat α = 0.001 of Kolmogorov-Smirnov (K-S) analysis for fluorescenceintensities of PI-stained T cells as the sigmoidal function of theaccumulated cell frequency curve and fluorescence intensity (channelnumber) compared to the curve of PI-stained T cells from the mediumcontrol (asterisk) as computed by Cellquest software (BD Biosciences)(Young, I. T. (1977) J. Histochem. Cytochem., 25: 935-941). ^(e)Index ofsimilarity = D/[(n_(c) + n_(t))/(n_(c) · n_(t))]^(1/2), where n_(c) andn_(t) are the number of events in cell frequency curves for mediumcontrol (c) and test substance (t), respectively (BD Biosciences)(Young, I. T. (1977) J. Histochem. Cytochem., 25: 935-941). An index ofsimilarity = 0 indicates the curves are identical.

N-4YSyn-Stimulated Immune SPC Enhance Dopaminergic Cell Death

To directly test the neurotoxic capacity of the N-4YSyn immune response,proliferating T cells or supernatants from N-4YSyn stimulated SPC ofN-4YSyn immunized mice were assessed in live/dead assays withco-cultures of N-α-Syn activated bone marrow-derived macrophages (BMM)and MES 23.5 cells. Minimal cytotoxicity was afforded from co-culturesof unstimulated BMM/MES 23.5 co-cultures with virtual 100% MES 23.5 cellsurvival, whereas activation with aggregated N-α-Syn resulted in 7% celldeath after 24 hours (FIG. 11). Activated and proliferating T cells from5 day cultures of antigen-specific stimulation of N-4YSyn immune SPCwhen added to activated BMM/MES 23.5 cultures induced 21% cell death(N-4YSyn+SPC), while supernatants (Sup) from those SPC cultures resultedin 44% cell death (N-4YSyn+SPC Sup), and no cytotoxicity to activatedBMM cultured in the absence of MES 23.5 cells (Macrophages+SPC Sup). InTranswell™ studies wherein N-4YSyn-stimulated BMM was separated from MES23.5 targets, virtually all cytotoxicity was concentrated among the MES23.5 cell population (MES 23.5 Transwell™) with no cytotoxicityattributed to BMM. T cells isolated from N-4YSyn-immunized donors andrestimulated in vitro with N-4Ysyn, but not 4YSyn, showed TNF-α levels(26.0±1.2 pg/ml) that were increased (p<0.008) compared to levels from Tcells of either PBS or 4YSyn immunized mice which were below the limitsof detection.

Example 2

Parkinson's disease (PD) is characterized by progressive nigrostriataldegeneration and deficits in dopamine transmission. A pathologicalhallmark of PD are Lewy bodies (LB) that present as intracellularinclusions of aggregated proteins and lipids in dopaminergic (DA)neurons (Duda et al. (2000) Am. J. Pathol., 157:1439-1445; Hurtig et al.(2000) Neurology 54:1916-1921). A major constituent of LB is α-synuclein(α-syn; Giasson et al. (2000) Science 290:985-989), characterized byself-aggregation and covalently bonded protein dimers modified byoxidative stress and protein nitration (Krishnan et al. (2003)Biochemistry 42:829-837; Paxinou et al. (2001) J. Neurosci.21:8053-8061; Souza et al. (2000) J. Biol. Chem., 275: 18344-18349).

Linkages between DA neurodegeneration and microglial neuroinflammatoryactivities are well-known and demonstrated by large numbers ofimmune-competent microglia within the substantia nigra of postmortem PDbrains appearing as phagocytic cells engulfing damaged DA neurons(McGeer et al. (1988) Neurology 38:1285-1291; Cho et al. (2003) Cell.Mol. Neurobiol., 23:551-560). In transgenic mutant α-syn mice and inmice treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)and rotenone (Cho et al. (2003) Cell. Mol. Neurobiol., 23:551-560;Kohutnicka et al. (1998) Immunopharmacology 39:167-180; Sugama et al.(2003) Brain Res., 964:288-294), similar microglial responses areoperative. Importantly, such microglial activation is associated withα-syn deposition (Jin et al. (2007) J. Neuroinflammation 4:2; Ling etal. (2004) Exp. Neurol., 190:373-383; Luo et al. (2007) Int. J. Mol.Med., 19:517-521) and internalization (Liu et al. (2007) J. ProteomeRes., 6:3614-3627). This occurs throughout disease, suggesting linkagesto oxidative damage, α-syn nitration and aggregation, and PD-associatedneurodegeneration (McGeer et al. (2004) Parkinsonism Relat. Disord., 10:S3-S7; Teismann et al. (2003) Proc. Natl. Acad. Sci., 100:5473-5478).Indeed, microglial activation is strongly associated with neurotoxicresponses and collateral neuronal damage (Gao et al. (2008) Environ.Health Perspect., 116: 593-598; Huang et al. (2008) Neurosci. Bull.,24:66-72; Stefanova et al. (2007) Mov. Disord., 22:2196-2203; Xiao etal. (2007) J. Neurochem., 102:2008-2019). Thus, for PD, nitrated-α-syn(N-α-syn)-mediated microglial activation and accelerated neuronal deathare closely related (Benner et al. (2008) PLoS ONE 3:e1376; Hodara etal. (2004) J. Biol. Chem., 279:47746-47753; Reynolds et al. (2008) J.Neurochem., 104: 1504-1525; Reynolds et al. (2008) J. NeuroimmunePharmacol., 3: 59-74; Thomas et al. (2007) J. Neurochem., 100:503-519).

Abundant evidence indicates a significant role for adaptive immunity inneuroregulatory activities (Benner et al. (2004) Proc. Natl. Acad. Sci.,101:9435-9440; Garg et al. (2008) J. Immunol., 180:3866-3873; Laurie etal. (2007) J. Neuroimmunol., 183: 60-68; Reynolds et al. (2007) J.Leukocyte Biol., 82:1083-1094). Such effects are seen in experimentalneurodegenerative models, including PD (Reynolds et al. (2007) J.Leukocyte Biol., 82:1083-1094; Gorantla et al. (2008) Glia 56:223-232;Banerjee et al. (2008) PLoS ONE 3:e2740). Principally, these data foundthat neuronal degeneration or protection is linked to the microglialphenotype and that N-α-syn-specific effector T cells exacerbatemicroglial activation and DA neurodegeneration, whereas CD4+ CD25+regulatory T cells (Treg) attenuate those processes; however, howmicroglia activation is regulated by regulatory and effector T cellsubsets is not known. To address this, aggregated N-α-syn was used as aninducer of microglial activation (Reynolds et al. (2008) J. Neurochem.,104:1504-1525; Reynolds et al. (2008) J. Neuroimmune Pharmacol.,3:59-74), then studied the microglial immune response as it is affectedby activated Treg and CD4+ CD25+ effector T cells (Teff). Theobservations demonstrate, for the first time, that Treg modulate a broadrange of microglial activities, including redox biology, migration,phagocytosis, energy metabolism, and cytokine secretions. Differentialoutcomes of microglial processes are dependent on the temporalengagement of Treg with N-α-syn and microglia. The findings provideinsights into disease pathobiology and how the adaptive immune systemmay be harnessed for therapeutic benefit.

Materials and Methods Animals

C57BL/6J male mice (7 wk old) were purchased from The Jackson Laboratoryand used for CD4+ T cell isolations. All animal procedures were inaccordance with National Institutes of Health guidelines and wereapproved by the Institutional Animal Care and Use Committee of theUniversity of Nebraska Medical Center.

Cell Isolates

Microglia were prepared from neonatal mice (1-2 days old) usingpreviously described techniques (Dobrenis et al. (1998) Methods16:320-344). Adherent microglia were cultured in DMEM complete mediumfor 7-14 days and then replated for experiments. Cultures wereconsistently >98% CD11b+ microglia as determined by morphology and flowcytometric analysis (Enose et al. (2005) Glia 51:161-172). CD4+ T cellsubsets were isolated from lymph nodes and spleens using previouslydescribed techniques (Reynolds et al. (2007) J. Leukocyte Biol., 82:1083-1094; Banerjee et al. (2008) PLoS ONE 3:e2740). Teff and Tregisolates used in these studies were >95% enriched. Following CD3activation, T cells were added in coculture with primary microglia for24 hours. All analyses of microglia phenotypic changes were performedafter removal of T cells by vigorous washings from the microglialcocultures.

Flow Cell Analysis

Samples from cell fractions were labeled with fluorescently labeled Absto CD4, CD8, CD25, CTLA-4, CD62L, Fas ligand (FasL), Fas (APO-1), CD11b,and intracellular FoxP3 (eBioscience) and active caspase-3 (Abcam) andanalyzed with a FACSCalibur flow cytometer (BD Biosciences). FITC-latexbeads (1 mM, 2.5% solids; Sigma-Aldrich) were added to microglialcultures for 30 minutes. Microglia were then detached and acid-washed(PBS, pH 6.0) to quench fluorescence of nonphagocytosed beads, and cellswere analyzed by flow cytometry and gated to CD11b+ cells. Fluorescenceintensity was normalized to beads alone.

Quantitative PCR (qPCR)

RNA was extracted with TRIzol® reagent (Invitrogen), column-purified(Qiagen), and RNA (2 μg) was reverse-transcribed with random hexamersand SSII reverse transcriptase (Applied Biosystems) for cDNA synthesis.Real-time qPCR was performed with cDNA using an ABI PRISM 7000 sequencedetector (Applied Biosystems), using the SYBR Green detection system andmurine-specific primers. Values were normalized to Gapdh expression.

Recombinant α-syn

Purification, nitration, and aggregation of recombinant mouse α-syn wereperformed as described previously (Reynolds et al. (2008) J. Neurochem.,104:1504-1525; Thomas et al. (2007) J. Neurochem., 100:503-519; Reynoldset al. (2007) J. Leukocyte Biol., 82:1083-1094). N-α-syn was added tocultures at 100 nM (14.5 ng/ml).

Cytokine/Chemokine Analysis

Fifty microliters of culture supernatants were analyzed using the BDCytometric Bead Array Mouse Inflammation kit (BD Biosciences) andmeasured with a FACSCalibur flow cytometer (BD Biosciences). Cytokineconcentration was determined from a standard curve prepared fromcytokine standards. The multianalyte cytokine ELISArrays (Superarray)were performed according to the manufacturer's protocol. Culture inserts(0.4-μm pore size; BD Biosciences) and neutralizing Abs to mouse IL-10(5 μg/ml; BD Pharmingen), TGF-β1 (5 μg/ml; R&D Systems), and CTLA-4(CD152, 5 μg/ml; BD Biosciences) were used.

2D SDS-PAGE

Cell lysate fractionation, sample labeling, 2D DIGE, image acquisition,and Decyder analysis were performed as described previously. Theselection criteria for spots were based on gel image quantitativeanalysis using DeCyder software (GE Healthcare) with the threshold foranalysis at >1.5-fold difference between spot intensities. Protein spotswere analyzed by biological variance analysis (BVA) software (GEHealthcare), then matched to a preparative 2D gel and excised using anEttan robotic spot picker (Rozek et al. (2007) J. Proteome Res.6:4189-4199). In-gel tryptic digestion and LC-MS/MS were performed asdescribed previously. Proteins identified by peptides having a UnifiedScore >3000 were targeted for further analysis (Enose et al. (2005) Glia51:161-172).

Western Blot Analysis

Ten micrograms of protein were loaded onto 4-12% gradient Bis-TrisNuPAGE® Novex gels (Invitrogen), electrophoresed, and transferred ontopolyvinylidene difluoride membranes (Bio-Rad). Blots were probed withthe respective primary Abs and secondary Abs (1/10,000; Invitrogen) andwere detected using a Super-Signal West Pico Chemiluminescent substrate(Pierce). Band intensity was measured using ImageJ and normalized toGapdh or β-actin (1/5000; Santa Cruz Biotechnology).

Immunofluorescence

Intracellular reactive oxygen species (ROS) production was detectedusing the ImageItLive ROS Detection kit (Invitrogen) according to themanufacturer's protocol. Abs included active caspase-3 (Abcam) and NF-κBp65 (Cell Signaling Technology), and nuclei were stained with TO-PRO-3iodide or 4′,6-diamidino-2-phenylindole (DAPI; Invitrogen). Images weretaken with a Nikon swept field confocal microscope (Nikon). Cathepsin B(CB) activity was determined using the CV-Cathepsin B Detection kit(BIOMOL) according to the manufacturer's protocol and was visualizedwith an inverted fluorescent microscope. The mean fluorescence intensity(MFI) was determined using ImageJ software.

Glutathione (GSH) Assay

Microglia were cultured with and without N-α-syn for 24 hours in mediawithout exogenous glutamine. Intracellular GSH levels were determinedwith the Biovision GSH Assay kit (Biovision) according to themanufacturer's protocol, and assessed using a SpectraMAX GEMINIfluorometer (Molecular Devices) at excitation/emission of 340/450 nm andnormalized to a GSH standard curve.

Apoptosis

Apoptotic cells were detected using the TACS TdT Fluorescein In SituApoptosis Detection kit (R&D Systems) according to the manufacturer'sprotocol and were visualized by a fluorescent microscope. MFI of TUNEL+cells was determined per field using ImageJ and was normalized toDAPI-stained nuclei (n=3, six fields per well). Caspase activity wasdetermined using the SensoLyte Homogeneous R110 Caspase-3/7 Assay kit(AnaSpec) according to the manufacturer's protocol. Cell viability wasdetermined by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoliumbromide (MTT) activity as described previously. Functional grade Abs tomouse FasL (2 μg/ml; eBioscience) and Fas (5 μg/ml; BD PharMingen) andCA074ME (BIOMOL) were used.

Statistics

All values are expressed as means±SEM and are representative of three tofour separate experiments. Differences among means were analyzed byone-way ANOVA, followed by Tukey's post-hoc testing for pairwisecomparison. For identification of statistically significant proteins,three to four analytical gels were analyzed using BVA software byone-way ANOVA for pairwise comparison between treatment groups.

Results

Treg Affect N-α-Syn microglial NF-κB Responses

To test the notion of Treg control of microglial activities inpreclinical and overt disease, two experimental paradigms weredeveloped. One reflects early or asymptomatic disease where Treg wouldengage microglia before exposure to N-α-syn and the second, where Tregare added to N-α-syn-activated microglia. Tests of cell surface Ags,cytokine gene expression, and suppression of Teff proliferationindicated that T cell isolates were characteristic of distinct Treg andTeff populations. To determine the effect of CD4+ T cells on microglialresponses to N-α-syn, CD3-activated Treg or Teff were cocultured withprimary microglia at a 1:1 ratio for 24 hours, removed the T cells, andstimulated the microglia with aggregated N-α-syn. Microglial uptake ofCy5-labeled N-α-syn by flow cytometry for Cy5-N-α-syn-containingmicroglia between control and T cell-treated microglia revealed thatneither Treg nor Teff treatment significantly altered microglia uptakeof N-α-syn. In situ analysis for NF-κB p65 expression in culturedmicroglia revealed that N-α-syn stimulation resulted in an increase inNF-κB p65 expression compared with unstimulated controls. In contrast,pretreatment with Treg, but not Teff, attenuated the induction of NF-κBp65 expression by N-α-syn stimulation (FIG. 12A). Western blot analysisfor NF-κB activation was determined by translocation of the subunitsRELA/p50 and NFκB1/p65 to the nucleus. N-α-syn stimulation inducedtranslocation of the NF-κB subunits to the nucleus, whereastranslocation was inhibited by pretreatment with Treg (FIG. 12B). AfterTeff pretreatment, translocation of NF-κB subunits was comparable toN-κ-syn stimulation. Diminished expression of NF-κB-related genesfollowing pretreatment with Treg in stimulated microglia, includingTnfa, Tnfrs1a, Re1a, and Nos2, was also observed (FIG. 12C). Expressionof neurotrophins Bdnf and Gdnf was increased following Treg pretreatmentto greater levels relative to all other treatments (FIG. 12D).

Microglial cytokine/chemokine analysis revealed that pretreatment withTreg suppressed production of IFN-γ, TNF-α, IL-12, IL-6, IL-10, andMCP-1 compared with untreated or Teff-treated microglia, whereas onlyTNF-α was reduced by Teff pretreatment (FIG. 13A). To mirrorinteractions that would occur between CD4+ T cells and microglia indisease, microglia were first stimulated with aggregated N-α-syn for 12hours before addition of Treg or Teff (post-treatment). Tregposttreatment resulted in diminished production of all assayedproinflammatory cytokines, except IFN-γ, which was increased as a resultof Treg or Teff coculture compared with N-α-syn stimulation alone,although concentration was less after treatment with Treg than withTeff. Assessment of CD206 (macrophage mannose receptor) and MHC class IIexpression revealed that pre- or posttreatment with Treg up-regulatedboth markers for alternative activation, as did posttreatment with Teff(FIGS. 13B and 13C). N-α-syn stimulation reduced microglial phagocytosisof FITC-labeled latex beads as determined by a 68% decrease in MFIcompared with unstimulated microglia (FIG. 13D). However,Treg-pretreated microglia consistently engulfed more beads compared withN-α-syn-stimulated microglia (+6.8-fold) or Teff-pretreated microglia(+1.9-fold). In contrast, post-treatment with Treg or Teff had nosignificant effect on phagocytosis compared with N-α-syn stimulation.

Treg-Modulated Microglial Responses Require Factor Secretion and CellContact

To determine whether Treg-mediated attenuation of microglialinflammation depends on T cell-microglia contact or on cytokine support,microglia were cocultured with Treg either in Transwell™ or withneutralizing Abs to IL-10, TGF-β, or CTLA-4. After 24 hours, inserts,Abs, and Treg were removed, and the microglia were stimulated withN-α-syn for 24 hours. Inhibition of IFN-γ secretion was not affected byphysical contact but was abrogated or reduced in the presence ofneutralizing Ab to IL-10 and TGF-β, respectively (FIG. 13E). Suppressionof TNF-α was partially reversed in the presence of neutralizing Abs forIL-10, TGF-β, or CTLA-4 but not in Transwell™ cultures, whereasinhibition of IL-12 production was dependent on both IL-10 and TGF-β. Incontrast, inhibition of MCP-1 was seen in Transwell™ cultures or withneutralizing Ab against IL-10, TGF-β, or CTLA-4, suggesting that bothcell contact and soluble factors attenuate MCP-1 production. Suppressionof IL-1α was dependent, in part, on cell contact and TGF-β, whereasinhibition of IL-1α was IL-1α dependent (FIG. 13F). Modulation ofphagocytic activity of microglia was primarily dependent on cell contactand was reduced 12-fold in Transwells™ compared with coculture. Neitherblockade of IL-10 nor CTLA-4 altered phagocytic function compared withcoculture; however, inhibition of TGF-β resulted in a 2-fold reductionin FITC-gated cells compared with coculture without Ab (p<0.05).

Treg and the Microglial Proteome

To facilitate quantitative detection and to maximize identification ofchanges in the microglial proteome in response to N-α-syn followingcoculture with Treg, microglial cell lysates were subjected to 2D gelelectrophoresis and LC-MS/MS proteomic analyses. Representativeanalytical 2D gels and Decyder™ analyses are shown for cell lysates ofN-α-syn-stimulated microglia compared with unstimulated controls (FIG.14A). In comparison to N-α-syn-stimulated microglia, coculture with Tregbefore stimulation resulted in a different proteomic profile (FIG. 14B),as did coculture with Treg post-activation (FIG. 14C). Analysis wasperformed on analytical gels from separate lysates comparing microgliacultures stimulated with media alone, N-α-syn, or cocultured with Tregby BVA software to identify differentially expressed proteins (p≦0.05).Proteomic analyses of N-α-syn/Teff cocultures vs N-α-syn stimulationalone following pre- and posttreatment were also performed to facilitatecross-comparisons between treatments by BVA. All analytical gels werecross-compared by BVA and matched to a preparative gel consisting ofpooled protein from the experimental groups. Identified spots werecompared for area and peak height (3D plots) by BVA. Western blotanalyses and densitometry confirmed differential expression of severalproteins, including L-plastin (+1.5-fold), ferritin L chain (+1.3-fold)and peroxiredoxin 1 (+1.5-fold), and cathepsin D (−2.0-fold) inmicroglial lysates following pretreatment with Treg compared withstimulated with N-α-syn alone (FIG. 14D).

Among the proteomic changes induced by pretreatment of microglia withTreg and compared with N-α-syn stimulated microglia were decreasedexpression in several cytoskeletal proteins such as β-actin, vimentin,cofilin 1, and gelsolin, whose function is to regulate cell motility andvesicle transport. Treatment with Treg also resulted in increasedexpression of microglial proteins involved in exocytosis such as annexinA1 and annexin A4, and phagocytosis such as L-plastin. Stimulation withN-α-syn decreased expression of proteins associated with theubiquitin-proteasome system (UPS)>1.5-fold compared with unstimulatedmicroglia, whereas pretreatment with Treg increased expression ofUPS-related proteins, including proteosome subunit α type 2, proteasomesubunit β type 2, ubiquitin specific protease 19, and ubiquitin fusiondegradation. Treatment with Treg also increased the expression ofmolecular chaperones, including heat shock proteins (HSP) andcalreticulin; most of which were decreased following stimulation withN-α-syn compared with unstimulated controls. Lysosomal proteases,including cathepsins B and D, were increased by N-α-syn stimulationalone; however, microglia pretreated with Treg showed decreasedabundance of the same proteins. Regulatory proteins involved in cellularmetabolism (transaldolase 1) and catabolism (α-mannosidase) wereincreased in Treg-pretreated cultures.

Changes in several proteins associated with mitochondrial function wereobserved as a result of stimulation with N-α-syn. Of interest, proteinsof the electron transport chain (ETC), specifically complex V, involvedin ATP synthesis were decreased in expression. Whereas ETC proteins suchas nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) Fe—Sprotein 2 of complex I, cytochrome c oxidase of complex III, and thesubunits that make up the components of ATP synthase were increased bymicroglia in response to N-α-syn stimulation following Tregpretreatment. Changes in the mitochondrial response to Treg were notlimited to proteins involved in cellular energetics but included redoxproteins, chaperones, and structural proteins. Other proteins thatincreased as a result of treatment with Treg were mitochondrial redoxproteins, including peroxiredoxins, superoxide dismutase (SOD)2,thioredoxin 1 (THX 1), and catalase. In addition, cytoplasmic redoxproteins were also increased, including peroxiredoxin 1, SOD1,biliverdin reductase B (BVR B), and glutaredoxin 1 (GLU 1).Interestingly, all were decreased by N-α-syn stimulation compared withunstimulated controls.

For comparison of the microglial phenotype after commitment toactivation by N-α-syn stimulation and modulation by CD3-activated Tcells, microglia were first stimulated with N-α-syn for 12 hours beforethe addition of Treg or Teff for an additional 24 hours, and the T cellswere removed before microglial cell lysis. Similar proteins wereaffected by post-treatment with Treg as with pretreatment;interestingly, some exhibited opposite expression patterns observedafter pretreatment with Treg. Western blot analysis and densitometryconfirmed differential expression of L-plastin (−1.6 fold), ferritin Lchain (+1.2-fold), peroxiredoxin 1 (+1.5-fold), and cathepsin D(+1.5-fold) by posttreatment with Treg compared with N-α-syn alone (FIG.14D).

Akin to pre-treatment, post-treatment with Treg yielded increasedredox-active protein expression by activated microglia, includingSOD1and peroxiredoxins 1 and 5. Several proteins that weredifferentially expressed in the pretreatment analysis were alsoidentified in post-treatment analysis but were opposite in direction,including increased expression of structural proteins involved in cellmotility, such as β-actin and γ-actin, decreased expression ofmitochondrial proteins, including ETC complex V, and decreasedexpression in L-plastin. Induction of proapoptotic protein expressionwas observed, including increased expression of apoptosis-associatedspeck-like protein containing a caspase recruitment domain, galectin 3,gelsolin, eukaryotic translation elongation factor 1, and cathepsins Band D. Decreased expression of proteins involved in cellular metabolismsuch as aldolase I and aldehyde dehydrogenase 2 was also observed inresponse to Treg post-treatment.

Treg Affect Microglial Oxidative Stress

To validate that changes in expression of redox-active proteinsaccurately reflect changes in the oxidative balance of microglia,oxidative stress levels were measured in N-α-syn-activated microgliapretreated with Treg or Teff. N-α-syn-stimulated microglia consistentlyproduced greater levels of H₂O₂ compared with unstimulated controls,whereas Treg pretreatment of microglia diminished the levels of H₂O₂(FIGS. 15A and 15B). In contrast, pretreatment with Teff exacerbatedH₂O₂ production by microglia. Analysis of intracellular GSH levelsrevealed that microglia stimulated with N-α-syn were depleted ofintracellular GSH, a key oxidative buffer in cells, following 24 hoursof stimulation as previously shown (Reynolds et al. (2008) J.Neuroimmune Pharmacol., 3:59-74). However, pretreatment with Tregbuffered the loss of GSH stores in stimulated microglia, whereas Teffprovided no significant protection from GSH loss (FIG. 15C). Westernblot and densitometric analyses validated protein expression trendsidentified by proteomics for select redox-active proteins, including THX1 (+1.7-fold; FIG. 15D), biliverdin reductase B (BVR B) (+1.8-fold; FIG.15E), HSP 70 (+1.4-fold; FIG. 15F), and GLU 1 (+2.0-fold; FIG. 15G).

Treg Modulate Microglial CB Activity

Treg pretreatment revealed decreased expression of cellular proteases,including cathepsin B (CB). Therefore, it was investigated whetherdifferential protein expression paralleled inhibition of CB enzymaticactivity. N-α-syn stimulation of microglia for 24 hours increased CBactivity compared with unstimulated controls, whereas pretreatment withTreg before stimulation diminished CB activity (FIGS. 15H and 15I). Incontrast, stimulated microglia pretreated with Teff exhibited CBactivity similar to that of N-α-syn stimulation alone. Western blotanalysis revealed increased abundance of CB both in cell lysates andculture supernatants of N-α-syn-stimulated microglia compared withunstimulated controls and cultures pretreated with either Treg or Teff,whereas pretreatment with Treg diminished intracellular (−1.6-fold) andsecreted (−1.8-fold) CB levels (FIG. 15J).

Proapoptotic Treg Responses are Mediated Through Fas-FasL Interactions

Evidence that Treg regulate inflammation through induction of apoptosisin activated effector cells, including monocytes/macrophages (Glanzer etal. (2007) J. Neurochem., 102:627-645; Liu et al. (2009) J. Immunol.,182:3856-3866), led to the investigation of whether post-treatment withTreg induced microglial apoptosis. Microglial cell viability wasmonitored using independent markers for apoptosis and cell viability:caspase-3 activation, MTT activity, and TUNEL. By Western blot analysis,lysates from microglia cultured for 24 hours in the presence of N-α-synexhibited increased caspase-3 activation compared with unstimulatedcontrols (FIG. 16A). Pretreatment with Treg or Teff resulted indiminished caspase-3 activation in response to N-α-syn, whereasposttreatment with Treg or Teff increased levels of cleaved caspase-3products. To confirm those results, analysis of active caspase-3 by flowcytometry revealed that pretreatment of microglia with either Treg orTeff failed to significantly increase caspase-3 activation (FIG. 16B).In contrast, post-treatment with Treg, but not Teff, resulted in asignificant increase in active caspase-3+ cells. In situ staining foractive caspase-3+ cells revealed that posttreatment with Treg resultedin profound induction of active caspase-3 relative to any othertreatment paradigm (FIG. 16C).

To investigate the proapoptotic factors involved in the Treg effect onmicroglia, the relative expression of FasL on Treg and Teff was assessedby flow cytometry after fresh isolation (naive), anti-CD3 activation, orafter coculture with N-α-syn-stimulated microglia. Numbers of FasL+Tregand Teff were increased following anti-CD3 activation (FIG. 5D). Incomparison, coculture with N-α-syn-activated microglia induced >80% ofTreg to express FasL, whereas microglial coculture had no significantadditive effect on Teff. N-α-syn activation diminished Fas (CD95)expression by microglia; however, coculture of microglia with eitheractivated T cell subset resulted in significant up-regulation of Fasexpression among activated microglia (FIG. 16E). Moreover, reducedexpression of Fas by N-α-syn-stimulated microglia paralleled reducedsusceptibility to anti-CD95-induced apoptosis compared with unstimulatedcontrols (FIG. 16F). Although pretreatment with Treg did not reducemicroglial viability, pretreatment with Treg, but not Teff, restoredsusceptibility of N-α-syn-stimulated microglia to anti-CD95-inducedapoptosis. In contrast, post-treatment with T cells resulted insignificant apoptosis of microglia compared with unstimulated andN-α-syn-stimulated microglia, with Treg inducing >2-fold increase in MFIof TUNEL+microglia (FIG. 16G). T cell-mediated apoptosis ofN-α-syn-stimulated microglia was mediated through Fas-FasL interactionsas anti-FasL returned levels of TUNEL staining to those ofN-α-syn-stimulated controls. These results were essentially confirmed byMTT assays of microglia, showing reduction of microglial cell viabilityafter posttreatment with Treg and Teff and increased viability afterblocking of Treg with anti-FasL (FIG. 16H). This apoptotic response wasat least partially caspase-dependent as Treg and Teff posttreatmentincreased activation of caspase-3/7 in N-α-syn-stimulated microgliacompared with controls, whereas incubation with anti-FasL partiallyblocked caspase activation (FIG. 16I).

Analysis of the N-α-syn microglial proteome following post-treatmentwith Treg revealed increased abundance of CB in cell lysates comparedwith N-α-syn stimulation alone. Treg-induced expression of CB wasvalidated by Western blot and densitometry analyses (+1.6-fold; FIG.17A). The role for CB activation in Treg-mediated microglial apoptosiswas investigated using the cell-permeable inhibitor of CB, CA-074ME. Insitu staining for active caspase-3+ cells revealed that inhibition of CBpartially diminished active caspase-3 expression following stimulationwith N-α-syn (FIG. 17B). In comparison, CB inhibition during Tregpost-treatment resulted in significant suppression of active caspase-3expression relative to Treg post-treatment without CB inhibitor.Decreased active caspase-3 was also observed in Teff-treated cultures inresponse to CB inhibitor. Inhibition of CB partially inhibited loss ofTreg-mediated N-α-syn microglial MTT activity but had no significantaffect on Teff-mediated cytotoxicity (FIG. 17C). Similarly, inhibitionof CB diminished caspase-3/7 activation in N-α-syn-stimulated microgliatreated in the presence or absence of Treg or Teff (FIG. 6D). Analysisby flow cytometry confirmed these observations as inhibition of CBdiminished active caspase-3+ cells on average by 11.1±0.5% inN-α-syn-stimulated cultures. Moreover, flow cytometric analysis revealedthat neutralization of FasL during posttreatment resulted in decreasedCB protein expression in Treg-treated microglia by 40.2±4.0% (p<0.05compared with N-α-syn/Treg posttreatment), whereas blocking of FasLinteractions produced no significant effect on diminishing CB expressionin Teff-treated cultures (4.8±1.7%). Taken together, these data supporta role for the Fas-FasL proapoptotic pathway and the induction of CB topromote apoptosis in the effect of Treg poststimulation on activatedmicroglia.

Example 3

Parkinson's disease (PD) is a progressive neurodegenerative diseasecharacterized clinically as gait and motor disturbances such asrigidity, resting tremor, slowness of voluntary movement, and posturalinstability. In some cases these evolve to frank dementia (Dauer et al.(2003) Neuron 39:889-909; Fahn et al. (2000) In Merritt's Neurology,Rowland, L. P., Ed. Lippincott Williams & Wilkins: New York, pp 679-693;Fahn, et al. (2004) NeuroRx 1:139-54; Mayeux, R. (2003) Annu. Rev.Neurosci., 26:81-104). A plethora of host and environmental factorsaffect the onset and progression of PD including genetics, environmentalcues, aging, peripheral immunity, impaired energy metabolism, andoxidative stress (Baba et al. (2005) Parkinsonism Relat. Disord.,11:493-8; Klockgether, T. (2004) Cell Tissue Res., 318:115-20; Linton etal. (2004) Nat. Immunol., 5:133-9; Naylor et al. (2005) J. Immunol.,174:7446-52; Orr et al. (2005) Brain 128:2665-74; Reale et al. (2009)Brain Behav. Immun., 23:55-63; Rosenkranz et al. (2007) J.Neuroimmunol., 188:117-27; Sian et al. (1994) Ann. Neurol., 36:348-55;Taki et al. (2000) Eur. J. Nucl. Med., 27:566-73; Tanner, C. M. (1992)Occup. Med., 7:503-13; Tanner, C. M. (1992) Neurol. Clin., 10:317-29).Pathologically, PD is characterized by nigrostriatal degenerationprecipitated by progressive loss of dopaminergic neuronal cell bodies inthe substantia nigra pars compacta (SNpc) and their projections to thedorsal striatum (Homykiewicz et al. (1987) Adv. Neurol., 45:19-34). Thisdegeneration is associated with alterations in innate, microglialactivation and adaptive T cell immunity (Baba et al. (2005) ParkinsonismRelat. Disord., 11:493-8; Banati et al. (1998) Mov. Disord., 13:221-7;Block et al. (2004) Faseb J., 18:1618-20; Cicchetti et al. (2002) Eur.J. Neurosci., 15:991-8; Formo et al. (1992) Prog. Brain Res.,94:429-436; Hong, J. S. (2005) Ann. NY Acad. Sci., 1053:151-2; McGeer etal. (1998) Alzheimer Dis. Assoc. Disord., 12:S1-6; Mirza et al. (2000)Neuroscience 95:425-432; Wang et al. (2005) Mech. Ageing Dev.,126:1241-54; Benner et al. (2008) PLoS ONE 3:e1376; Brochard et al.(2009) J. Clin. Invest., 119:182-92; Theodore et al. (2008) J.Neuropathol. Exp. Neurol., 67:1149-58). Precipitation of immunedysfunction in PD is thought to ensue from the release of cytoplasmicinclusions of fibrillar, misfolded proteins encased in Lewy bodies (LB)and composed principally of aggregated α-synuclein (α-syn) (Spillantiniet al. (1997) Nature 388:839-40). Such misfolded proteins can engageinnate and adaptive immunity (Spillantini et al. (1997) Nature388:839-40; Croisier et al. (2005) J. Neuroinflammation 2:14). Indeed,substantive evidence supports the notion that nigrostriatal degenerationis manifest by α-syn mediated microglial activation, oxidative stressand disease inciting adaptive immune responses (Benner et al. (2008)PLoS ONE 3:e1376; Brochard et al. (2009) J. Clin. Invest., 119:182-92;Theodore et al. (2008) J. Neuropathol. Exp. Neurol., 67:1149-58;Reynolds et al. (2008) J. Neurochem., 104:1504-25; Reynolds et al.(2008) J. Neuroimmune Pharmacol., 3:59-74; Thomas et al. (2007) J.Neurochem., 100:503-19; Zhang et al. (2005) Faseb J., 19:533-42). It isthe pathogenic spiral of dopaminergic neuronal death, release ofextracellular aggregated α-syn, microglial activation, peripheral immuneactivation, collateral neuronal injury, sustained α-syn release withingress into lymphatics, and engagement of specific T cell responsesthat further damage dopamine neurons.

It has been demonstrated that microglia associated degenerativeresponses are triggered by nitrated α-syn (N-α-syn)-specific effector Tcells (Teff); whereas, CD4+ CD25+ regulatory T cells (Treg) attenuatemicroglial activation and promote dopaminergic neuronal survival(Reynolds et al. (2007) J. Leukoc. Biol., 82:1083-94). Lacking from theprior works was a mechanism for CD4+ T cell-mediated modulation ofmicroglial function. Based on these observations, it was hypothesizedthat CD4+ T cells have dual roles, and as such, influence microglialresponses to evoke biological activities that ultimately effect neuronalsurvival or loss. In attempts to decipher the mechanisms underlying suchresponses, aggregated N-α-syn was used as an inducer of microglialactivation (Reynolds et al. (2008) J. Neurochem., 104:1504-25; Reynoldset al. (2008) J. Neuroimmune Pharmacol., 3:59-74; Thomas et al. (2007)J. Neurochem., 100:503-19), then examined the microglial proteomeaffected by interactions with CD4+ T cell subsets (Reynolds et al.(2009) J. Immunol., 182:4137-49). Using proteomic approaches, it isdemonstrated that Treg regulatory activities extend beyond inhibition ofcellular activation and include modulation of a broad range ofmicroglial activities involving regulation of phagocytosis andproteasome function, induction of redox-active and bioenergeticproteins, and apoptotic cell processes. Such regulatory events lead tothe attenuation of microglial inflammatory neurotoxic responses.Importantly, the data demonstrate that the effects of Treg onN-α-syn-mediated immune activities are multifaceted and of potentialtherapeutic benefit.

Materials and Methods Animals

C57BL/6J male mice (7 weeks old) were purchased from The JacksonLaboratory (Bar Harbor, Me.) and used for CD4+ T cell isolations.C57BL/6J neonates were obtained from breeder colonies housed in theUniversity of Nebraska Medical Center animal facilities. All animalprocedures were in accordance with National Institutes of Healthguidelines and were approved by the Institutional Animal Care and UseCommittee of the University of Nebraska Medical Center.

Cell Isolates

Microglia were prepared from neonatal mice (1-2 days old) usingpreviously described techniques (Dobrenis, K. (1998) Methods 16:320-44).Cultures were consistently >98% CD11b+ microglia (Enose et al. (2005)Glia 51:161-72). CD4+ T cell subsets were isolated using previouslydescribed techniques (Reynolds et al. (2007) J. Leukoc. Biol.,82:1083-94; Banerjee et al. (2008) PLoS ONE 3:e2740). Treg and Teffisolates were >95% enriched (Reynolds et al. (2009) J. Immunol.,182:4137-49). CD3-activated T cells were co-cultured with microglia at1:1 ratio. All analyses of microglia were performed after removal of theT cells from the cultures.

Recombinant α-syn

Purification, nitration and aggregation of recombinant mouse α-syn wereperformed as previously described (Reynolds et al. (2008) J. Neurochem.,104:1504-25; Reynolds et al. (2008) J. Neuroimmune Pharmacol., 3:59-74;Thomas et al. (2007) J. Neurochem., 100:503-19). N-α-syn was added tocultures at 100 nmol/L (14.5 ng/ml).

2D Difference Gel Electrophoresis (DIGE) and Image Analysis

Protein prepared from microglial cell lysates was labeled with therespective CyDyes, followed by separation in the first and seconddimension, and the gels were scanned using a Typhoon 9400 Variable ModeImager. Analyses of Cy3-Cy5 image pairs, adjustment to Cy2 controlimages and detection of protein spots were performed using DeCyder™software (GE Healthcare). Statistical significance (P<0.05) wasdetermined with Biological Variance Analysis (BVA).

Mass Spectrometry

In gel trypsin digestion were performed as previously described(Ciborowski et al. (2004) J. Neuroimmunol., 157:11-6). The resultingpeptides were sequenced using Electrospray Ionization-LiquidChromatography Mass Spectrometry (ESI-LC MS/MS) (Proteome X System withLCQDecaPlus mass spectrometer, ThermoElectron, Inc.) with a nanosprayconfiguration. The spectra were searched against the NCBI.fasta proteindatabase narrowed to murine proteins using SEQUEST search engine(BioWorks 3.1 SR software from ThermoElectron, Inc.). Validation ofselect proteins identified by LC-MS/MS was performed usingimmunocytochemistry or Western blot.

Cytotoxicity

The Live/Dead Viability/Cytotoxicity kit (Invitrogen) was performedaccording to manufacturer's protocol. Images were taken usingfluorescence microscopy. Cell counts were normalized as the percentageof surviving cells from unstimulated culture controls.

Statistics

For identification of statistically significant proteins, three-to-fouranalytical gels were analyzed using BVA software by one-way ANOVA forpair-wise comparison between treatment groups. Differences betweenmeans±SEM were analyzed by one-way ANOVA followed by Tukey's post-hoctest for pair-wise comparisons.

Results Microglial Protein Profiling Techniques Following N-α-SynStimulation and Treg Co-Cultivation

It has been demonstrated that aggregated N-α-syn induces activation ofthe NF-κB pathway in microglia resulting in a robust inflammatoryresponse characterized by increased production of TNF-α, IFN-γ, IL-6,and IL-1β among others (Reynolds et al. (2008) J. Neurochem.,1041504-25; Reynolds et al. (2008) J. Neuroimmune Pharmacol., 3:59-74).Co-culture of microglia with Treg either pre- or post-stimulationsignificantly attenuates NF-κB activation as well as inflammatorycytokine and superoxide production in response to N-α-syn, whereas Teffexacerbate these responses (Reynolds et al. (2007) J. Leukoc. Biol.,82:1083-94; Reynolds et al. (2009) J. Immunol., 182:4137-49). Therefore,to uncover putative mechanisms for CD4+ T cell-mediated modulation ofthe microglial phenotype, 2D DIGE was used to identify differences inprotein expression of N-α-syn stimulated of microglia alone andcocultured with CD4+ T cells. 2D DIGE analysis of microglial celllysates was repeated three separate times with three independent cellisolations and cultures. Analyses of 2D images from protein lysates of15×10⁶ microglial cells identified an average of approximately 2000“putative” protein spots. DeCyder™ DIGE Analysis of Cy3-labeled proteinsfrom unstimulated microglia and Cy5-labeled proteins from N-α-synstimulated microglia obtained from three independent experiments showedan average of 2072 detected spots. Representative analyses revealed 43%differentially expressed protein spots after setting a threshold mode ofquantitative differences ≧2 standard deviations (SD). Of those uniquelyidentifiable spots (582), 28% were upregulated and (318) 15% weredownregulated in microglial cell lysates in response to 24 hourstimulation with N-α-syn. To assess how CD4+ T cells modulate theN-α-syn microglial phenotype, microglia were co-cultured with eitherTreg or Teff for 24 hours either prior to stimulation with N-α-syn(pre-treatment) or following 12 hours of stimulation (post-treatment),and comparisons were made using 2D DIGE and nano-LC-MS/MS peptidesequencing (FIG. 18). Co-cultivation with Treg prior to stimulation withN-α-syn (pre-treatment) altered the microglial phenotypic response toN-α-syn stimulation. An analysis of Cy3-labeled proteins from N-α-synstimulated microglia and Cy5-labeled proteins from N-α-syn stimulatedmicroglia pre-treated with Treg obtained from three independentexperiments showed an average of 2326 detected spots. Representativeanalysis revealed 31% differentially expressed protein spots aftersetting a threshold mode of quantitative differences ≧2 SD. Of thoseuniquely identifiable spots (348), 15% were increased and (365) 16% weredecreased in microglial cell lysates in response to Treg treatment priorto N-α-syn stimulation. Pre-treatment with Teff had less robust affectson the microglial phenotype in response to N-α-syn. Of the >2000uniquely identifiable spots, approximately 32 (1.8%) were decreased and22 (1.3%) increased in abundance compared to N-α-syn stimulation alone.

To mimic what may occur during overt disease, CD4+ T cells were added toN-α-syn microglial cultures 12 hours post-stimulation. Co-cultivationwith Treg post-stimulation with N-α-syn (post-treatment) also alteredthe microglial phenotype. An analysis of Cy3-labeled proteins fromN-α-syn stimulated microglia and Cy5-labled proteins from N-α-synstimulated microglia post-treated with Treg obtained from threeindependent experiments showed an average of 1905 detected spots.Representative analysis revealed 27% differentially expressed proteinspots after setting a threshold mode of quantitative differences ≧2 SD.Of those uniquely identifiable spots, (110) 6% were increased and (403)21% were decreased in microglial cell lysates in response to Tregtreatment following N-α-syn stimulation. By comparison, post-treatmentwith Teff resulted in significant modulation of the microglial proteomein response to N-α-syn stimulation. Of the >2000 uniquely identifiablespots, approximately 318 (15%) were decreased and 325 (16%) wereincreased in abundance compared to N-α-syn stimulation alone.

To identify differentially expressed proteins (P<0.05), analyses withBVA software were performed on analytical gels from separate lysatescomparing microglia cultures stimulated with media alone, N-α-syn orco-cultured with Treg or Teff to facilitate cross-comparisons betweentreatments by BVA whereby identified spots were compared for area andpeak height (3D plots). The 3D peak of each protein spot, comprised ofCy3-labeled and Cy5-labeled cell lysates, was generated based on thepixel intensity versus pixel area, where peak area correlated with thedistribution of the protein spot on the gel. 3D images were obtainedusing 2D Master Imager and were evaluated independently based on theirdifferential fluorescent signal within a constant area for the spot.Their relative peak volumes were normalized to the total volume of thespot (Cy2-labeled). All analytical gels were cross-compared by BVA andmatched to a preparative gel consisting of pooled protein from theexperimental groups. The proteins identified consisted of structural orcytoskeletal proteins, regulatory proteins, redox-active proteins andenzymes. FIG. 19 shows the location of these proteins on the preparativegel selected for LC-MS/MS sequencing.

N-α-syn Stimulation and the Microglial Proteome

It has been demonstrated that N-α-syn is capable of inducing thetemporal activation of a neurotoxic microglial phenotype (Reynolds etal. (2008) J. Neurochem., 104:1504-25; Reynolds et al. (2008) J.Neuroimmune Pharmacol., 3:59-74). To extend these works, the time courseof activation was extended from 2 hours, 4 hours, and 8 hours to 24hours for the current study. FIGS. 22A-22G shows proteins differentiallyexpressed in microglia that were stimulated in media alone or withN-α-syn. Proteins were considered identified with high confidence withat least two peptides sequenced and met the threshold peptide criteria.Such threshold criteria have been determined previously to result in a95% confidence level in peptide identification (Ciborowski et al. (2007)Virology 363:198-209; Ricardo-Dukelow et al. (2007) J. Neuroimmunol.,185:37-46). The categories of proteins included regulatory,cytoskeleton/structural, enzymes, mitochondrial, redox-active andothers. FIG. 20A shows the relative percentages of proteins within eachclassification based on protein function that were modulated by N-α-synstimulation and expression trends.

A majority of the proteins positively identified by mass spectrometrywere decreased in expression. A large percentage of the proteins thatwere decreased in response to N-α-syn stimulation following 24 hourswere cytoskeletal associated including vimentin, cofilin 1, beta-actinand alpha-tubulin (FIGS. 22A-22G). N-α-syn stimulation also resulted indecreased expression of proteins involved in protein processing,transport, and folding. These included cryptochrome 2, 14-3-3 zeta, andannexin A1, as well as several molecular chaperones including heat shockprotein (Hsp) 10, Hsp 60, and Hsp 70. Moreover, stimulation with N-α-syndecreased expression of proteins associated with theubiquitin-proteasome system (UPS) greater than 1.5-fold compared tounstimulated microglia (FIGS. 22A-22G). Several proteins associated withmitochondrial function and redox biology were also decreased as a resultof stimulation with N-α-syn. Of interest, proteins of the electrontransport chain (ETC), specifically complex V involved in adenosinetriphosphate (ATP) synthesis, were decreased in expression. Redox-activeproteins were also decreased following 24 hours of exposure to N-α-synincluding superoxide dismutase (Sod)1, biliverdin reductase B,peroxiredoxin (Prdx) 1 and glutaredoxin 1 (FIGS. 22A-22G). Otherproteins decreased following stimulation with N-α-syn stimulation weremetabolic proteins such as acetylcoenzyme A and aldehyde dehydrogenase,and proteins involved in glycolysis such as alpha enolase, pyruvatedehydrogenase, and pyruvate kinase (FIGS. 22A-22G). Despite theeven-distribution of up- and down-regulated proteins identified in theinitial analysis, many of the proteins that were increased in expressiondid not reach the confidence interval threshold for adequateidentification by mass spectrometry. Nonetheless, those identifiedincluded lysosomal proteases cathepsins B and D, gelsolin implicated ininflammation and proteins involved in catabolism including aldo-ketoreductase family 1 member B8 and catechol o-methyltransferase (FIGS.22A-22G).

Treg-Microglial Co-Cultivation Followed by N-α-Syn Stimulation(Pre-Treatment)

To simulate preclinical disease and assess putative mechanisms for earlyaffects of CD4+ T cells on the microglial phenotype in response toN-α-syn, microglial cells were co-cultured with CD3-activated CD4+ Tcells for 24 hours prior to exposure to N-α-syn. FIGS. 22H-22M showthose proteins differentially expressed in microglia stimulated withN-α-syn alone or pre-treated with Treg. The relative percentages ofproteins within each classification based on protein function that weremodulated by Treg pretreatment together with N-α-syn stimulation andexpression trends are shown in FIG. 20B. Among the proteomic changesinduced by pre-treatment of microglia with Treg prior to N-α-synstimulation were decreased expression in several cytoskeletal proteinssuch as β-actin, vimentin, cofilin 1, and gelsolin, involved inregulation of cell motility and vesicle transport. Treatment with Tregalso resulted in increased expression of microglial proteins involved inexocytosis such as annexin A1 and annexin A4, and phagocytosis such asL-plastin (FIGS. 22H-22M). In addition, pre-treatment with Tregincreased expression of UPS-related proteins including proteasomesubunit alpha type-2, proteasome subunit beta type-2, ubiquitin specificprotease 19 and ubiquitin fusion degradation. Treatment with Treg alsoincreased the expression of molecular chaperones including HSPs andcalreticulin. Whereas lysosomal proteases cathepsins B and D wereincreased by N-α-syn stimulation alone, microglia pre-treated with Tregshowed decreased abundance of the same proteins. Regulatory proteinsinvolved in cellular metabolism (transaldolase 1) and catabolism(α-mannosidase) were increased in Treg pre-treated cultures (FIGS.22H-22M).

ETC proteins such as nicotinamide adenine dinucleotide (NADH)dehydrogenase (ubiquinone) Fe—S protein-2 of complex I, cytochrome coxidase of complex III and the subunits that comprise the components ofATP synthase were increased by microglia in response to N-α-synstimulation following Treg pre-treatment. Changes in the mitochondrialresponse to Treg were not limited to proteins involved in cellularenergetic, but included redox proteins, chaperones, and structuralproteins. Other proteins increased as a result of pre-treatment withTreg were mitochondrial redox-active proteins including peroxiredoxins,Sod 1, Sod 2, thioredoxin (Thrx) 1 and catalase. In addition,cytoplasmic redox-active proteins were also increased including Prdx 1,biliverdin reductase B and glutaredoxin 1 (FIGS. 22H-22M).

Cross-comparison of Teff pre-treatments was facilitated by the BVAmodule to compare protein expression trends. In contrast topre-treatment with Treg, pre-treatment with Teff did not alter theexpression of structural proteins including cofilin 1 and 2, taxilinalpha or beta actin in response to N-α-syn stimulation. Expression oflysomal proteases including cathepsin B and D were also not changed. Inaddition, pre-treatment with Teff did not affect expression ofredox-active proteins such as Prdx 5, cytochrome c reductase, Thrx 1, orbiliverdin reductase B. However, enzymatic proteins that were involvedin glycolysis and metabolism were decreased in expression following Teffpre-treatment included pyruvate kinase M, phosphoglycerate kinase andaldolase A. Proteins of the ETC were also decreased including ATPsynthase (Complex V). Compared to N-α-syn stimulation alone, Teffpretreatment resulted in greater than 1.5 fold increased expression ofvoltage-dependent anion channel-1 (Vdac-1), the interferon α/β receptor,and Prdx 1, whereas Hsp 90, chaperonin, galectin 3 and gelsolin weredecreased greater than 1.5 fold in expression.

N-α-syn Stimulation Followed by Treg-Microglial Co-Cultivation(Post-Treatment)

For comparison of the microglial phenotype after commitment toactivation by N-α-syn stimulation and modulation by CD3-activated CD4+ Tcells, microglia were first stimulated with N-α-syn for 12 hours priorto the addition of Treg or Teff for an additional 24 hours and the Tcells removed prior to microglial cell lysis. FIGS. 22N-22Q show thoseproteins differentially expressed in microglia stimulated with N-α-synalone or post-treated with Treg. The relative percentages of proteinswithin each classification based on protein function that were modulatedby Treg post-treatment together with N-α-syn stimulation and expressiontrends is shown in FIG. 20C.

Similar proteins were affected by post-treatment with Treg as withpre-treatment; interestingly, some exhibited opposite expressionpatterns observed after pre-treatment with Treg. Akin to pre-treatment,post-treatment with Treg yielded increased redox-active proteinexpression by activated microglia including Sod1 and Prdx1 and 5.Several proteins differentially expressed in the pre-treatment analysiswere also identified in post-treatment analysis, but were expressed inopposite directions, including increased expression of structuralproteins involved in cell motility, such as β-actin and γ-actin,decreased expression of mitochondrial proteins including ETC complex V,and decreased expression of L-plastin (FIGS. 22N-22Q). Induction ofpro-apoptotic protein expression was observed and included increasedexpression of apoptosis-associated speck-like protein containing acaspase recruitment domain, gelsolin, eukaryotic translation elongationfactor 1, and cathepsins B and D. Decreased expression of proteinsinvolved in cellular metabolism such as aldolase I and aldehydedehydrogenase 2 was also observed in response to Treg post-treatment(FIGS. 22N-22Q).

Cross-comparison of protein expression trends following post-treatmentwith Teff revealed that in contrast to pre-treatment, post-treatmentwith Teff increased expression of redox-active proteins including Prdx1, Thrx 1, and cytochrome c oxidase in N-α-syn stimulated microgliacompared to N-α-syn stimulation alone. Ferritin light chain, Hsp 70, andtransaldolase 1 were also increased. Similar to pre-treatment,expression of cathepsins B and D were not affected. Moreover, expressionof proapoptotic proteins was not affected with Teff post-treatment.

Validation of Protein Identification and Biological Significance

Immunocytochemistry and Western blot analyses were used to validateprotein expression trends identified in the proteomic analyses.Immunoflourescent cytochemistry revealed that stimulation with N-α-synsignificantly reduced Prx1 expression in microglial cells compared tounstimulated microglia. In contrast, Treg pre-treatment protectedagainst a decrease in Prx1 expression (FIG. 21A). In comparison,post-treatment with Treg rescued microglial Prx1 expression and restoredexpression levels to near 100% of the unstimulated control. The effectof Teff was more variable and depended on the temporal engagement ofTeff with stimulated microglia. Pre-treatment with Teff did noteffectively alter Prx1 expression in response to N-α-syn stimulation,however Prx1 expression appeared to be partially rescued followingpost-treatment with Teff although this did not reach statisticalsignificance.

Western blot validation for cytoskeletal and inflammatory proteins thatwere involved both in cell mobilization as well as survival, confirmedexpression trends of select proteins following different cultureconditions (FIG. 21B). Expression of alpha-tubulin was decreased nearly6-fold following Treg pre-treatment, and compared to a 1.5 fold increaseby N-α-syn stimulation alone. In comparison, alpha tubulin expression inN-α-syn-stimulated microglia following Teff pre-treatment was reduced by2-fold. Post-treatment with Treg or Teff failed to alter alpha-tubulinexpression levels in N-α-syn stimulated microglia. Analysis of gelsolinconfirmed the increased expression in N-α-syn stimulated microgliallysates compared to control (1.5 fold). Pre-treatment with Treg reducedgelsolin expression to control levels, while, post-treatment increasedgelsolin expression compared to N-α-syn stimulation alone. Albeitpre-treatment with Treg had no effect on galectin 3 expression,post-treatment with Treg resulted in a 1.4 fold increase compared toN-α-syn stimulation alone. No change in expression of gelsolin orgalectin 3 was detected in response to Teff treatment by Western blot.

Immunofluorescence cytochemistry for actin and Hsp70 also confirmeddifferential expression of these proteins following N-α-syn stimulationand pre-treatment with CD4+ T cells. Whereas pretreatment with Tregsignificantly decreased fluorescence intensity of beta-actin expressionin response to N-α-syn stimulation, expression of Hsp70 was increasedcompared with N-α-syn stimulation alone to levels and exceeded thoseobserved in unstimulated controls. By comparison, pre-treatment withTeff had no observed affect on either actin or Hsp70 expression comparedwith N-α-syn stimulation alone (FIG. 21C).

Deleterious microglial activation is postulated to affect aneurodegenerative process in PD. For this reason, suppression ofmicroglial activation by Treg may be responsible for the profoundprotection observed in vivo (Reynolds et al. (2007) J. Leukoc. Biol.,82:1083-94). To investigate whether phenotypic modulation of microgliaby Treg co-culture affected neuronal survival, an in vitro model ofmicroglia-mediated cytotoxicity was established using N-α-syn-activatedmicroglia and the dopaminergic cell line MES23.5. A 56% loss of MES23.5cells was observed after co-culture for 24 hours with N-α-syn stimulatedmicroglia compared to control cocultures of MES23.5 with unstimulatedmicroglia (FIG. 21D). In contrast, co-culture of N-α-syn stimulatedmicroglia with Treg inhibited microglial-mediated MES23.5 cytotoxicity,while activated Teff afforded no cytotoxic protection. These datasuggested that Treg modulation of microglia attenuates theneurocytotoxic responses mediated by activated microglia. In addition,supernatants from microglia stimulated with N-α-syn alone or N-α-syn andcultured in the presence of Teff were cytotoxic to MES23.5 cells,whereas neurocytotoxicity was abrogated in supernatants from stimulatedmicroglia co-cultured with Treg. Surprisingly, there was lesscytotoxicity induced from culture supernatants from N-α-syn microgliatreated with Teff than seen in supernatants from N-α-syn microgliaalone. These data demonstrate the potential of Tregs to suppresscytotoxicity afforded by N-α-syn-activated microglia, and suggest thatdirect modulation of microglial responses provides a primary mechanismfor Treg-mediated neuronal protection.

Example 4

Parkinson's disease (PD) is a progressive neurodegenerative disordercharacterized by resting tremor, rigidity, bradykinesia, and gaitdisturbances (Fahn et al. (1998) Mov. Disord., 13:759-767; Mayeux, R.(2003) Annu. Rev. Neurosci., 26:81-104; Fahn et al. (2004) NeuroRx.,1:139-154). Presently, 1.5 million Americans are afflicted. Diseaseincidence rises with increasing age, with 120/100 000 contracting PDover the age of 70 (Dauer et al. (2003) Neuron 39:889-909).Pathologically, PD is characterized by the progressive loss ofdopaminergic neuronal cell bodies in the substantia nigra pars compacta(SNpc) and their termini in the dorsal striatum (Hornykiewicz et al.(1987) Adv. Neurol., 45:19-34). These pathological findings commonlyparallel microglial activation observed in association with deposits ofaggregated alpha synuclein (α-syn) in intracellular inclusions, known asLewy bodies (LB) (Spillantini et al. (1997) Nature 388:839-840; Croisieret al. (2005) J. Neuroinflammation 2:14). Although host genetics andenvironmental factors affect the onset and progression of PD (Tanner, C.M. (1992) Occup. Med., 7:503-513) significant clinical, epidemiologic,and experimental data also support a role for microglial inflammation indisease pathogenesis (Formo et al. (1992) Prog. Brain Res., 94:429-436;Banati et al. (1998) Mov. Disord., 13:221-227; McGeer et al. (1998)Alzheimer Dis. Assoc. Disord., 12(Suppl. 2):S1-S6; Mirza et al. (2000)Neuroscience, 95:425-432; Cicchetti et al. (2002) Eur. J. Neurosci.,15:991-998; Block et al. (2005) Prog. Neurobiol., 76:77-98; Hong, J. S.(2005) Ann. NY Acad. Sci., 1053:151-152; Wang et al. (2005) Mech. AgeingDev., 126:1241-1254).

The mechanisms underlying microglial activation in PD and how it affectsneuronal survival is incompletely understood. One line of investigationis that neuronal death itself drives microglial immune responses(Giasson et al. (2000) Science 290:985-989; Przedborski et al. (2001) J.Neurochem., 76:637-640; Mandel et al. (2005) Ann. NY Acad. Sci.,1053:356-375). Alternatively, it has been proposed that activationoccurs as a consequence of release of aggregated proteins from thecytosol or within LB to the extracellular space. In this way, the deathof dopaminergic neurons leads to release of modified protein aggregatesthat activate microglia inciting a lethal cascade of neuroinflammationand neuronal demise (Zhang et al. (2005) FASEB J., 19:533-542; Wersingeret al. (2006) Curr. Med. Chem., 13:591-602). Several lines ofexperimental evidence support this contention (Spillantini et al. (1997)Nature 388:839-840; Goedert, M. (1999) Philos. Trans. R. Soc. Lond. BBiol. Sci., 354:1101-1118; Giasson et al. (2000) Science 290:985-989;Kakimura et al. (2001) Eur. J. Pharmacol., 417:59-67; Croisier et al.(2005) J. Neuroinflammation 2:14; Lee et al. (2005) J. Neurosci.25:6016-6024). First, aberrant expression of α-syn and PD pathogenesisare linked. This is derived from the discovery that mutations andmultiple copies of the gene encoding α-syn (SNCA and PARK1) are linkedto familial early onset PD (Kruger et al. (1998) Nat. Genet.,18:106-108; Spira et al. (2001) Ann. Neurol., 49:313-319; Zarranz et al.(2004) Ann. Neurol., 55:164-173; Singleton et al. (2003) Science302:841; Chartier-Harlin et al. (2004) Lancet 364:1167-1169). Second,oxidation and nitration of α-syn leads to formation of aggregates andfilaments that comprise LB (Giasson et al. (2000) Science 290, 985-989;Souza et al. (2000) J. Biol. Chem., 275:18344-18349). Third, portions ofα-syn are secreted rendering it more vulnerable to aggregation (Lee etal. (2005) J. Neurosci., 25:6016-6024) and oxidative modification(Kakimura et al. (2001) Eur. J. Pharmacol., 417:59-67). Fourth, α-synitself can activate microglia, inducing reactive oxygen species (ROS)(Thomas et al. (2007) J. Neurochem., 100:503-519) and subsequentneurotoxicity (Zhang et al. (2005) FASEB J., 19:533-542). Fifth,microglial products including cytokines, chemokines, excitotoxins, andproteins of the classical complement cascade affect a broad range ofneurological diseases (McGeer et al. (1998) Alzheimer Dis. Assoc.Disord., 12(Suppl. 2):S1-S6; Bal-Price et al. (2001) J. Neurosci.,21:6480-6491; Liu et al. (2003) J. Pharmacol, Exp. Ther., 304:1-7; Blocket al. (2005) Prog. Neurobiol., 76:77-98). Sixth, endogenous activatorsof microglia show a neuroinflammatory fingerprint reflective of what canoccur during PD (Zhou et al. 2005; McLaughlin et al. 2006). Lastly,attenuation of microglial activation can protect up to 90% ofdopaminergic neurons in PD animal models (Du et al. (2001) Proc. Natl.Acad. Sci. USA, 98:14669-14674; Teismann et al. (2003) Proc. Natl. Acad.Sci. USA, 100:5473-5478; Wu et al. (2002) J. Neurosci., 22:1763-1771;Teismann et al. (2001) Synapse 39:167-174; Kurkowska-Jastrzebska et al.(2004) Int. Immunopharmacol., 4:1307-1318; Choi et al. (2005) J.Neurosci., 25:6594-6600; Vijitruth et al. (2006) J. Neuroinflammation3:6).

Based on these observations, changes in the microglial transcriptome andproteome as a consequence of the cells' engagement with nitrated andaggregated α-syn (N-α-syn) was investigated. N-α-syn stimulation ofmicroglia induced morphological cell transformation and neurotoxicsecretions. A N-α-syn-activated ‘microglial signature’ was determined bygene microarrays, 2D differential in-gel electrophoresis (DIGE), and bycytokine profiling. N-α-syn induced a microglia inflammatory phenotypecharacterized by the expression of neurotoxic and neuroregulatoryfactors. Importantly, the inflammatory signature seen in laboratoryassays were mirrored in parallel tests performed on postmortem braintissues from PD patients. These observations, taken together, indicate a‘putative’ role for N-α-syn-activated microglia in disease.

Materials and Methods Parkinson's Disease Brain Tissues

Autopsy materials from the substantia nigra (SN) and basal ganglia (BG;caudate nucleus and putamen) of 10 patients who died with signs andsymptoms of PD, three with Alzheimer's disease (AD), and 10 age-matchedcontrols were secured from the National Research Brain Bank TissueConsortium. The 10 controls ranged in age from 62 to 91 and died ofdiseases unrelated to neurological impairments. This includedatherosclerotic and metabolic diseases, infections, and cancer.

An antibody to N-α/β-syn (clone nSyn12, mouse ascites; Upstate,Charlottesville, Va.) that recognizes nitrated human N-α-syn (14.5 kDa)and N-α-syn (17 kDa) was used for immunoprecipitation. Samples of SNfrom control, AD, and PD autopsy brain tissues were homogenized inice-cold radioimmunoprecipitation (RIPA) buffer, pH 7.4 and centrifugedat 10 000 g for 10 minutes at 4° C. to remove cellular debris. ProteinA/G PLUS-agarose beads (Santa Cruz Biotechnology Inc., Santa Cruz,Calif., USA) were added to 1 mg total cellular protein and incubated for30 minutes at 4° C. Beads were pelleted by centrifugation at 1000 g for5 minutes at 4° C. The supernatants were incubated overnight at 4° C.with 2 μg of primary antibody, then with Protein A/G PLUS-agarose beadsfor 6 hours on a rotating device at 4° C. Immunoprecipitates (IP) werecollected after centrifugation at 1000 g for 5 minutes at 4° C., washedwith phosphate-buffered saline (PBS), and resuspended in 20 μL of 1×electrophoresis sample buffer.

Nitrated-α-Syn IP (20 μL) were fractionated by 16% Tricine sodiumdodecyl sulfate-polyacrylamide gel electrophoresis (PAGE) (Jule Inc.,Milford, Conn., USA and BIORAD Laboratories Inc., Los Angeles, Calif.,USA) at constant voltage for 1.5 hours. The gels were fixed and stainedwith Coomasie Blue to visualize protein bands. Bands corresponding tothe molecular weights encompassing 14.5 kDa (α-syn) were excised,digested by trypsin, column purified, and sequenced by liquidchromatography-tandem mass spectrometry (LC-MS/MS) for proteinvalidation. Sequenced peptides were distinguished by peptide matches tothe human α-syn sequence (NCBI Accession:AA108276).

Purification, Nitration, and Aggregation of Recombinant Mouse α-Syn

Purification, nitration, and aggregation of recombinant mouse α-syn wereperformed as previously described (Thomas et al. (2007) J. Neurochem.,100:503-519). Five individual lots of α-syn were tested for endotoxin byLimulus amebocyte lysate tests and all were below the limit of detectionfor endotoxin (<0.05 endotoxin units). Amino acid analysis to determineprotein concentration using HPLC was performed by the University ofNebraska Medical Center Protein Structure Core Facility. Proteins wereseparated by PAGE using 4-12% NuPAGE gels (Invitrogen, Carlsbad, Calif.,USA). After electrophoresis, the gels were transferred ontopolyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, Mass.,USA) and probed with primary mouse IgG1 anti-α-syn (1:500; TransductionLaboratories/BD Biosciences, Franklin Lakes, N.J., USA) or primaryrabbit IgG antinitrotyrosine (1: 2000; Upstate). Signal was detectedwith horseradish peroxidase-conjugated anti-mouse IgG or anti-rabbit IgG(both from Zymed Laboratories, South San Francisco, Calif., USA) usingchemiluminescence systems (SuperSignal® West Pico ChemiluminescentSubstrate; Pierce Biotechnology Inc., Rockford, Ill., USA). Forvisualization of the protein by atomic force microscopy (AFM), sampleswere deposited on mica, glued to a glass slide, and dried under argongas flow. The image was taken in air, height, amplitude, and phase modesusing a Molecular Force Probe 3D controller (Asylum Research Inc., SantaBarbara, Calif., USA).

Isolation, Cultivation, and N-α-syn Activation of Murine Microglia

Microglia from C57BL/6 mice neonates (1-to-2-days old) were preparedaccording to well described techniques (Dobrenis 1998). All animalprocedures were in accordance with National Institutes of Healthguidelines and were approved by the Institutional Animal Care and UseCommittee of the University of Nebraska Medical Center. Brains wereremoved and placed in Hanks' Balanced Salt Solution at 4° C. The mixedglial cells were cultured for 7 days in Dulbecco's Modified Eagle'sMedium (DMEM) containing 10% fetal bovine serum (FBS), 10 μg/mLgentamicin, and 2 μg/mL macrophage colony stimulating factor (WyethInc., Cambridge, Mass., USA). To obtain homogenous microglial cellpopulations, culture flasks were gently shaken and non-adherentmicroglia were transferred to new flasks. The flasks were incubated for30 minutes to allow the microglia to adhere, and loose cells removed bywashing with DMEM. Microglia were plated at 2×10⁶ cells per well insix-well plates in DMEM containing 10% FBS, 10 μg/mL gentamicin, and 2μg/mL macrophage colony stimulating factor. One week later, cells werestimulated with 100 nmol/L of aggregated N-α-syn/well or no stimulationfor 4 hours. Media were replaced with serum-free DMEM without phenol redor other additives (Invitrogen) and incubated for 24 hours in a 37° C.,5% CO₂ incubator.

Inflammatory Genomic and PCR Assays

RNA from N-α-syn stimulated primary murine microglial cells andunstimulated control was extracted with TRIzol® (Invitrogen), columnpurified (Qiagen, Valencia, Calif., USA), precipitated with ammoniumacetate, amplified and labeled using the T7-based TrueLabeling-AMP™ 2.0kit (Superarray, Frederick, Md., USA). The resultant cRNA was hybridizedto an oligo-based microarray for mouse general pathway (Superarray#OMM-014) and nuclear factor-kappa B (NF-κB)-related genes (Superarray#OMM-025). The arrays were washed, incubated sequentially withstreptavidin-bound alkaline phosphatase and chemiluminescent substratebefore exposure to X-ray film. Subsequent analysis of the microarrayswas performed using the GEArray expression analysis suite (Superarray).

Total RNA obtained from analysis of the microglial transcriptome wasreverse transcribed with random hexamers and SSII reverse transcriptase(Applied Biosystems, Foster City, Calif., USA). Murine-specific primerpairs were: Ccl2: CCCCAAGAAGGAATGGGTCC (SEQ ID NO: 3) andGGTTGTGGAAAAGGTAGTGG (SEQ ID NO: 4); I11b: GTTCCTTTGTGGCACTTGGT (SEQ IDNO: 5) and CTATGCTGCCTGCTCTTACTGACT (SEQ ID NO: 6); I110:CAGTTATTGTCTTCCCGGCTGTA (SEQ ID NO: 7) and CTATGCTGCCTGCTCTTACTGACT (SEQID NO: 8); Ifng: TTTGAGGTCAACAACAACCCACA (SEQ ID NO: 9) andCGCAATCACAGTCTTGGCTA (SEQ ID NO: 10); andNos2:5′-GGCAGCCTGTGAGACCTTTG-3′ (SEQ ID NO: 11) and5′-GAAGCGTTTCGGGATCTGAA-3′ (SEQ ID NO: 12). TaqMan® gene expressionassays specific for murine Tnf, Tnfrsf1a, Stat1, Rela, Bdnf, and Gdnfwere purchased from Applied Biosystems, and normalized toglyceraldehyde-3-phosphate dehydrogenase (Gapdh) expression. Tissuesamples obtained from PD and control patients were snap frozen on dryice and stored at −80° C. RNA was prepared from the samples usingTRIzol® reagent (Invitrogen) and purified with the RNeasy Mini Kit(Qiagen), prior to cDNA synthesis. Human specific primers for TNF,TNFRSF1A, STAT1, NFKB1, RELA, BDNF, and GDNF were analyzed using TaqMangene expression assays. Gene expression was normalized to thehousekeeping gene Gapdh. Real-time quantitative PCR was performed withcDNA using an ABI PRISM 7000 sequence detector (Applied Biosystems).Reverse SYBR Green I detection system was used, and the reactionsgenerated a melting temperature dissociation curve enabling quantitationof the PCR products.

Cytokine Arrays

Microglia were plated at a density of 2×10⁶ cells/well in a six-wellplate and stimulated with 100 nmol/L aggregated N-α-syn, and 100 ng/mLlipopolysaccharide (LPS, Escherichia coli; Sigma-Aldrich, St. Louis,Mo., USA) in serum-free media. Fifty microliter aliquots were collectedat 8, 24, and 72 hours of incubation in triplicate and frozen at −80° C.For assay, the samples were analyzed using the BD Cytometric Bead ArrayMouse Inflammation Kit (BD Biosciences, San Jose, Calif., USA) accordingto the manufacturer's protocol. Samples of culture supernatants frommicroglia were diluted 1:3 and 1:10 in assay diluent and analyzed forcytokine concentration with a FACSCalibur flow cytometer (BDBiosciences). Concentrations of cytokines were determined from astandard curve created with serial dilutions of the cytokine standardsprovided by the manufacturer.

Neurotoxicity Assays

MES23.5 cells were cultured in 75-cm² flasks in DMEM/F12 with 15 mmol/LHEPES (Invitrogen) containing N2 supplement (Invitrogen), 100 U/mL ofpenicillin, 100 μg/mL streptomycin, and 5% FBS. Cells were grown to 80%confluence then co-cultured at 1:1 ratio with previously platedmicroglial cells. To assess cell viability microglia cells were platedat a density of 5×10⁴ cells on sterile glass coverslips, and co-cultureswere prepared with a 1:1 ratio microglia: MES23.5 cells. After 24-48hours, cells were stimulated with aggregated 100 nmol/L N-α-syn or 100nmol/L α-syn for 4, 8, 24, and 72 hours. CD11b+ microglial cells weredistinguished from MES23.5 cells by APC-conjugated CD11b (1:200;Invitrogen) immunocytochemistry. For tyrosine hydroxylase (TH)cytostaining, cells were fixed in 4% p-formaldehyde, permeablized, andblocked in 2% normal goat serum with 0.25% Triton X-100 in PBS for 30minutes, and probed with rabbit polyclonal anti-TH (1:1000; EMDBiosciences Inc., San Diego, Calif., USA), followed by FITC goatanti-rabbit IgG. For western blot analysis, 10 lg of protein sample fromcell lysates of each treatment group was loaded onto a 12% NuPAGEBis-Tris gel (Invitrogen). Following transfer onto a PVDF membrane, themembrane was blocked and then probed overnight with anti-TH (1:1000).Signal was detected with horseradish peroxidase-conjugated anti-rabbitIgG (1:10 000; Zymed Laboratories) using chemiluminescence system(SuperSignal® West Pico Chemiluminescent substrate; Pierce BiotechnologyInc.). Densitometric analysis was performed using IMAGEJ software andnormalized to β-actin (1:1000; Abcam, Cambridge, Mass., USA). Assays forviable and dead mammalian cells (Live/Dead Viability/Cytotoxicity;Invitrogen) were performed according to manufacturer's protocol. Theprotocol was revised so that the concentration of each dye was 1 μmol/Lto avoid high background. Live cells were distinguished by the uptake ofcalcein acetoxymethyl ester to acquire a green fluorescence[excitation/emission (ex/em) 495/515 nm], while dead cells acquired ared fluorescence (ex/em 495/635 nm) because of the uptake of ethidiumhomodimer-1. Cell enumerations were performed using fluorescencemicroscopy (200· magnification) and a M5 microplate fluorometer(Molecular Devices, Sunnyvale, Calif., USA) (Limit 1 ex/em 490/522 nmand Limit 2 ex/em 530/645 nm). The number of viable MES23.5 cells ineach treatment group was normalized as the percentage of surviving cellsin unstimulated culture controls.

Protein Purification, 2D DIGE, and DeCyder Analyses

Cell lysates of microglia were prepared with 5 mmol/L Tris-HCl, pH 8.0,1% 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate and acocktail of protease inhibitors (Sigma-Aldrich). Protein content wasquantitated using a DC Protein Assay (BioRad, Hercules, Calif., USA).Factors known to interfere with isoelectric focusing (first dimensionseparation in 2D sodium dodecyl sulfate-PAGE) such as salts anddetergents were removed from cell lysates using the 2D Cleanup kit (GEHealthcare, Piscataway, N.J., USA) according to manufacturer's protocol.Protein concentration was determined using 2D Quant (GE Healthcare).Samples of control and stimulated cell lysates (25 μg of each lysate)were labeled with 400 pmol of CyDyeo 2. A 50 μg protein sample ofcontrol cell lysate was labeled with 400 μmol of CyDye 3; and a 50 μgprotein sample of stimulated cell lysate was labeled with 400 μmol ofCyDye 5. Labeling was performed following the manufacturer's protocols.The samples were pooled, resuspended in rehydration buffer to a totalvolume of 450 μL, then loaded onto an immobilized pH gradient strip, andleft for 18 hours for rehydration. In the first dimension, samples wererun in IPGphor and in Ettan DALTsix electrophoresis apparatus (GEHealthcare) for the second dimension. CyDye 3- and CyDye 5-derivatizedproteins were detected in gels using a Typhoon 9400 Variable Mode Imagerwith ex-em filters at 540/590 nm for CyDye 3 dyes and 620/680 nm forCyDye 5 dyes (GE Healthcare). Analysis of CyDye 3-CyDye 5 image pairs,adjustment to CyDye 2 control images, and detection of protein spotswith relative spot volumes were performed using DeCyder software (GEHealthcare) to locate and analyze multiplexed samples within the gel.Selected protein spots of interest were excised from the 2D gel using anEttan Spot Picker. The proteins from gel pieces were digested withtrypsin, as described below, and resultant peptides were analyzed usingLC-MS/MS system (ThermoElectron Inc., Waltham, Mass., USA). Proteinidentification was completed using BIOWORKS 3.1 software.

In Gel Tryptic Digestion and Protein Identification by LC-MS/MS

Specific protein spots were excised from the gels by an automated Ettanspot picker. Following column purification (ZipTip CU-18; Millipore)with 50% acetonitrile (ACN), 50 mmol/L NH₄HCO₃/50% ACN, and 10 mmol/LNH₄HCO₃/50% ACN, gel pieces were dried and incubated with trypsin (100ng/1L) (Promega, Sunnyvale, Calif., USA) for 12-18 hours. Samples wereextracted by 0.1% trifluoroacetic acid/60% ACN, pooled, and dried.

Dried peptide samples were reconstituted in 0.1% formic acid/HPLC-gradewater, detected on a ProteomeX LCQ™ DECA XP Plus mass spectrometer(ThermoElectron Inc.), and identified using BIOWORKS 3.1 SR software.Proteins identified by peptides having a Unified Score (BIOWORKS 3.1 SR,ThermoElectron Inc.) greater than 3000 were marked for further analysis.

Nuclear/Cytosol Fractionation

Cell lysates were prepared from SN of PD and control patients byhomogenization in PBS. Cells were collected following centrifugation at500 g for 5 minutes. Cytosol and nuclear fractions were prepared usingthe Nuclear/Cytosol Fractionation Kit (BioVision, Mountain View, Calif.,USA) according to manufacturer's protocol.

Western Blot Assays

Protein was prepared from cell lysates in RIPA buffer supplemented withprotease inhibitors (Pierce Biotechnology Inc.). Protease inhibitorcocktail was added to each conditioned media sample fraction prior toprocessing. Following centrifugation at 10 000 g for 10 minutes, thesupernatants were removed and allowed to dialyze against waterovernight. Tissue samples obtained from PD and control patients weresnap frozen on dry ice and stored at −80° C. Protein lysates wereprepared from individual samples through homogenization in RIPA buffersupplemented with protease inhibitors (Pierce Biotechnology Inc.).Protein quantification was performed using the bicinchoninic acid kit(Pierce Biotechnology Inc.). Protein concentration of each sample wasdetermined using a calibration curve generated from purified bovineserum albumin. A total of 20 μg of each sample was loaded onto 4-12%Bis-Tris NuPAGE gels (Invitrogen) and transferred onto PVDF membranes(BioRad). Primary antibodies to calmodulin (1:1000) and 14-3-3σ (1:200)(Millipore), biliverdin reductase (1:5000), thioredoxin (1:2000),β-actin (1:5000), and α-tubulin (1:5000) purchased from Abcam, L-plastin(1:1000), α-enolase (1:1000), glutathione-S-transferase (1:1000), andNF-κB p65 and p50 (1:200) purchased from Santa Cruz Biotechnology Inc.were used for analyses. Blots were probed with the respectivehorseradish peroxidase-conjugated secondary:antibodies (1:5000;Invitrogen) and detected using SuperSignal® West Pico Chemiluminescentsubstrate (Pierce Biotechnology Inc.). The intensity of protein bandswas quantified using IMAGEJ and normalized to Gapdh (1:5000; Santa CruzBiotechnology Inc.) level in the same sample.

Statistical Analyses

All values are expressed as mean±SEM. Differences among means wereanalyzed by Student's t-test or by one-way ANOVA followed by Bonferronipost hoc testing for pair-wise comparison.

Results Aggregated N-α-syn and Microglial Activation: Laboratory andPathological Studies

To investigate the mechanisms by which N-α-syn-mediated microglialactivation affects dopaminergic neurodegeneration, a cellular model wascreated that would reflect the salient features of neuroinflammation asit could occur in PD. To this end, it was first determined if N-α-synwas present in regions of brain where microglial activation is known tobe present in PD. Whole cell lysates consisted of several protein bandsfollowing gel electrophoresis and Coomassie staining. IP assaysperformed from SN tissues of PD patients using a primary antibodyagainst nitrated α/β-synulcein showed a greater than twofold increase inintensity of the protein band corresponding to 14-14.5 kDa (p<0.001)than that present in control patients (FIG. 23A) or in patientsdiagnosed with AD along with higher molecular weight species greaterthan 17 kDa. Peptide sequence analyses by LC-MS/MS revealed that theprotein band encompassing the 14-14.5 kDa of the anti-N-α-syn IPcontained peptides with sequence homology to human α-syn in SN samplesrecovered from PD brains (FIG. 23A, highlighted sequences).Interestingly, such sequence homologies to α-syn were not identifiedfrom 14 to 14.5 kDa proteins in either control or AD samples. Thus, anin vitro model was developed to reflect conditions present in anaffected human host, but using the murine analog. Here, recombinantmouse α-syn was purified, nitrated, and aggregated for use as amicroglial stimulant. Western blot assays showed cross-linking of N-asynmonomers (Souza et al. (2000) J. Biol. Chem., 275:18344-18349) andhigher molecular weight aggregates, thus verifying the nitration andaggregation of α-syn (FIG. 23B). The aggregated N-α-syn contained asubstantially reduced monomeric band (corresponding to a band at ˜14kDa) but higher molecular weight banding aggregates. Analysis of proteinaggregation was also assessed by AFM. Samples of N-α-syn contained lownumbers of globular aggregates (2-6 nm in height) prior to aggregation.However, following aggregation, N-α-syn was present predominately asoligomers (2-6 nm in height). In addition, there were few protofibrils(1.5-2.5 nm in height), filaments, and fibrils (˜5-8 nm in height)present (FIG. 23C). Non-nitrated α-syn was present in similarconfigurations.

The stimulatory effects of N-α-syn on microglia was then evaluated. Thedose of 100 nmol/L (14.5 ng protein/mL) was selected based on previousextensive works performed demonstrating that, following a dose-responseof N-α-syn, 100 nmol/L (50% over control) is required to inducesubstantive ROS from activated microglial cells (Zhang et al. (2005)FASEB J., 19:533-542; Thomas et al. (2007) J. Neurochem., 100:503-519.)as well as cytotoxicity. ROS production was slightly decreased incomparison with either 50 or 500 nmol/L of N-α-syn. While native α-synis ubiquitously expressed, the physiological concentration of N-α-syn indisease has not been elucidated. However, based on concentrations ofmodified α-syn in affected PD brain tissues, 100 nmol/L concentration isat physiologically relevant levels (Halliday et al. (2005) Brain 128,2654-2664) and is below that detectable by immunohistochemistry inneuronal inclusions within the SN of PD brains (≧100 ng). Phenotypictransformation into an ameboid morphology commonly follows microglialactivation with different pro-inflammatory stimuli (Giulian et al.(1995) J. Neurosci., 15:7712-7726; Vilhardt, F. (2005) Int. J. Biochem.Cell Biol., 37:17-21). Thus, it was examined if changes in microglialmorphology would be elicited following N-α-syn activation. Restingmicroglia were both round and ellipsoid shaped with retracted processesthat were characteristic of a relatively quiescent phenotype (FIG. 23D).In contrast, N-α-syn activated microglia assumed a more ameboidappearance with extensive processes, characteristic, in part, of anactivated phenotype. N-α-syn-stimulated microglia co-cultured withMES23.5 cells acquired a rod-like appearance and further extension ofprocesses.

It has been demonstrated that 100 nmol/L of aggregated N-α-syn couldactivate microglia to produce copious amounts of ROS (Thomas et al.(2007) J. Neurochem., 100:503-519). In contrast, unaggregated N-α-syn orminced neuronal membrane fractions failed to induce significant amountsof ROS above control levels. This suggested that the microglial responseto N-α-syn was specific and could not be elicited in response tounaggregated protein or by phagocytosis under the same conditions.Therefore, the extent of the neuroinflammatory phenotype induced byN-α-syn stimulation of microglia was assessed. Quantification of commoncytokines and chemokines that are secreted in response to inflammatorystimuli was performed by cytometric bead array. LPS-activated microgliaserved as a positive control. Stimulation with N-α-syn enhanced thesecretion of TNF-α, IL-6, MCP-1 (FIG. 23E), and IFN-γ compared withbasal levels observed in unstimulated microglia. These results areconsistent with the induction of an inflammatory microglial phenotypefollowing N-α-syn stimulation. The parallels between N-α-syn andLPS-induced cellular effects support a commonality for innate immuneresponses in disease and suggest that these pro-inflammatory processesmay be common among mononuclear phagocytes that recognize disparateactivators.

N-α-syn-Stimulated Microglia are Neurotoxic to MES23.5 DopaminergicCells

To determine the effect of N-α-syn-activated microglia on neuronalsurvival, the dopaminergic MES23.5 cell line was used as an indicatorfor cytotoxicity measurements by co-culture with stimulated andunstimulated microglia. MES23.5 cell death was determined by measuringimmunoreactivity for the rate-limiting enzyme in dopamine synthesis, TH,expressed by MES23.5 cells, and the Live/Dead cell assay. Duringstimulation with 100 nmol/L N-α-syn, the number of TH+ cells declined inthe stimulated cultures, resulting in a significant diminution inTH-immunoreactive cells (8 hours: 74.6% of control; 24 hours: 53.4% ofcontrol, p<0.01; 72 hours: 48.5% of control, n=6, p<0.01). Western blotanalysis confirmed this observation, as TH expression decreased in atime-dependent manner over the course of N-α-syn stimulation(TH+/β-actin ratio at 8 hours: 94.6% of control; 24 hours: 86.2% ofcontrol; 72 hours: 64.9% of control, p<0.01). Analysis of cell viabilitywith the Live/Dead cell assay demonstrated that stimulation of microgliawith 100 mmol/L of N-α-syn followed by MES23.5 co-culture resulted inremarkable reduction of viable cells with concomitant increase in deadMES23.5 cells; whereas, fewer dead cells were observed in co-cultureswith microglia stimulated with α-syn (non-nitrated) after 24 hours (FIG.24A). Percentage of MES23.5 cell survival was less in co-cultures withmicroglia stimulated with α-syn (83%) and N-α-syn (58%) compared withunstimulated controls (95%) at 72 hours (FIG. 24B). The more sensitivefluorometric analysis revealed as early as 24 hours after stimulation asimilar pattern of progressive decline in viable cells in the presenceof α-syn and N-α-syn stimulated microglia to 76% and 65% of controls at24 hours of stimulation, respectively (FIG. 24C). Moreover,N-α-syn-mediated cytotoxicity was restricted to MES23.5 cells, asstimulation of microglia in the absence of MES23.5 cells neitheraffected microglial survival (FIG. 24D) nor yielded a significantdifference in the number of dead CD11b+ cells between control andstimulated cultures. In addition, cytotoxicity of MES23.5 cells was notelicited with N-α-syn in the absence of microglia (FIG. 24D).Furthermore aggregation of N-α-syn was necessary for inducing microgliacytotoxicity (FIG. 24D). Importantly, a decrease in the cell survivalwas observed when microglia were stimulated with either aggregated α-syn(93%) or N-α-syn (86%) for 24 hours, and co-cultured with MES23.5 cellsin Transwell™ inserts, but not unaggregated protein. MES23.5 culturesincubated with supernatants obtained from microglia stimulated witheither α-syn or N-α-syn resulted in decreased cell survival (89% and84%, respectively) compared with supernatants from unstimulatedmicroglia (FIG. 24E).

NF-κB Gene Expression and Nuclear Translocation in Pd

NF-κB pathway activation is critical for the initiation of inflammatoryevents including the production of inflammatory cytokines and chemokineslinked to inflammation and microglial activation. Acquisition of such aninflammatory phenotype may begin with induction of gene products thatultimately leads to neurotoxic factor production, cell migration, andapoptosis. To determine the extent to which this pathway was operativein PD, the SN and BG of PD brains and controls (those withoutneurological disease) were analyzed for NF-κB-related genes as well asneurotrophin expression (FIG. 25). Increases, albeit modest, were seenin NFKB1 expression from samples of SN from PD patients compared withcontrols; whereas, no significant difference was observed for RELAexpression. However, an eightfold increase in TNF expression wasobserved in the SN and BG together with a twofold increase in expressionof its receptor TNFRSF1A. STAT1 was minimally decreased in PD brains.Similarly, analysis of AD brain tissues as a control forneuroinflammatory pathology also revealed a moderate induction of NF-κBtranscription factors NFκB1 and RELA, while TNF expression was increased40- and 10-fold in the SN and BG along with modest elevations of STAT1in AD brain tissues compared with controls. Based on these findings, itwas reasoned that a compensatory trophic mechanism could be operative inPD. Indeed, BDNF was shown to be increased greater than sixfold in theSN and twofold in the BG in PD. Consistent with recent observations(Backman et al. (2006) Mol. Cell. Endocrinol., 252:160-166), GDNF wasincreased greater than 10-fold in the BG but no significant changes wereobserved in the SN.

A recent investigation by immunofluorescence analysis of midbrainsections revealed a marked increase in expression of NF-κB p65 in the SNof PD patients compared with controls, which co-localized to CD11b+microglia in addition to affected neurons. In the current study,cytosolic and nuclear fractions were prepared from the lysates of SN ofPD and control brain tissues, and lysates analyzed for NF-jB proteinsubunits p50 and p65. Increased expression of NF-jB subunits in both thecytosolic fractions and nuclear fractions were observed in PD braintissues (FIG. 26). Phosphorylation of serine 536 (pS536) critical forRelA/p65 transcriptional activity was also increased in PD braintissues.

N-α-syn-Activated Microglia and the PD Transcriptome are Linked throughNF-κB

The increased expression of NF-κB transcription identified in the SN ofPD brains and the microglial response to N-α-syn stimulation that wereconsistent with inflammatory responses suggested that one majorsignaling pathway induced by N-α-syn involves NF-jB activation. Use of ageneral microarray confirmed that NF-κB expression was increased bystimulation with N-α-syn (FIG. 27A). Using NF-κB-focused microarrays(FIGS. 27B, 27D, and 27E), increased expression of genes encodingpro-inflammatory cytokines was shown, including Tnf, Cc12, I16, andI11b. Also induced were those genes encoding the NF-jB transcriptionfactor subunits, Nfkb1, Nfkb2, and Rela. In addition, N-α-syn inducedgenes involved in other pathways, particularly those of themitogen-activated pathway, as indicated by the induction of theimmediate early genes, Fos and Raf1. At 4 hours post-stimulation,expression of most NF-κB-related genes peaked. The majority of genesinduced at 1 hour remained elevated, with the addition of theapoptosis-regulatory genes Card10 and Casp8. The NF-κB inhibitor,Nfkbia, was also induced but may become apparent only after removal orclearance of the stimulus, as Ikbkb expression was also induced at thistime. Removal of N-α-syn from microglial cells after 4 hours ofstimulation reduced most NF-κB genes to pre-stimulatory levels. At 8 and16 hours following removal of N-α-syn from culture, severalapoptosis-regulatory genes (Card10, Card11, and Cflar) were induced aswell as genes for receptors of cell activation and NF-κB stimulationincluding Tnfrsf1a and Cd40. These results were similar but lesser inmagnitude than stimulation of microglia with LPS (FIGS. 27B and 27F-H).Consistent with microarray analyses, quantitative RT-PCR analyses ofTnf, I11b, and Ccl2 genes indicated very high levels of transcripts forthese cytokines during stimulation by N-α-syn (10-, 3097-, and 16-foldincreases, respectively) over pre-stimulatory levels (FIG. 27C).Verification of gene expression during stimulation of other, lessabundant, NF-κB-related genes were achieved, including Tnfrsf1a(6.2-fold increase), Stat1 (2.3-fold increase), and Rela (3.6-foldincrease). N-α-syn stimulation also increased expression of Nos2(inducible nitric oxide synthase) and Ifng, both regulated by NF-κBactivation. Expression of the neurotrophins Bdnf and Gdnf were alsoincreased following N-α-syn stimulation.

N-α-syn-Activated Microglial Proteome Shows a Reactive InflammatoryPhenotype

Analysis of the N-α-syn microglial transcriptome showed differentialgene regulation and induction of the NF-κB pathway, indicative of aninflammatory microglial phenotype. Activation of this pathway influencesdownstream expression of proteins involved in processes includinginflammation, immune regulation, survival, and proliferation. Proteinexpression obtained from cell lysates were analyzed following 2, 4, and8 hours of stimulation with 100 nmol/L N-α-syn to assess the translationof differences in gene induction to intracellular protein expression.Two-dimensional DIGE was used to compare protein expression profiles ofunstimulated microglia (control) and N-α-syn-stimulated microglia (FIG.28). A complete listing of all proteins identified by LCMS/MS iscontained within FIG. 27.

Stimulation with N-α-syn resulted in differential expression of severalproteins that are likely a consequence of NFκB-related signalingpathways (FIG. 27) as soon as 2 hours after stimulation. Many proteinsdifferentially expressed could be attributed to oxidative stress,including the down-regulation of aconitase as well as the up-regulationof peroxiredoxin-1, -4, -5, superoxide dismutase, and heat-shock protein70.

After 4 hours of N-α-syn-stimulation, proteins decreased includedseveral cytoskeletal proteins including β-actin, cofilin-1, profilin-1,tropomysin-3, and vimentin. The putative functions of other proteinsdecreased in N-α-syn-stimulated microglial lysates were found to beinvolved in cell adhesion and actin microfilament attachment to theplasma membrane (vinculin, coronin-1A, and adenylyl cyclase-associatedprotein 1), glycolysis and growth control (α-enolase), and migration(galectin 3 and macrophage migration inhibitory factor) (Walther et al.(2000) J. Neurosci. Res., 61:430-435; Chandrasekar et al. (2005) J. CellSci., 118:1461-1472). Annexin A3 is an inhibitor of phospholipase A2 anda promoter of apoptosis of inflammatory cells (Parente et al. (2004)Inflamm. Res., 53:125-132), and was also down-regulated. The antioxidantglutaredoxin-1 was also decreased in cell lysates compared withunstimulated controls (FIG. 27). Four of the proteins increased instimulated cell lysates affect intracellular calcium signaling, storage,and cell cycle regulation (swiprosin 1, calmodulin, calreticulum, andnucleophosmin 1) (Parente et al. (2004) Inflamm. Res., 53:125-132;Vuadens et al. (2004) Proteomics 4:2216-2220; Meini et al. (2006) Eur.J. Neurosci., 23:1690-1700).

By 8 hours, 73 proteins were differentially expressed. Thirty threeproteins were decreased including all cytoskeletal proteinsdown-regulated at 4 hours, vimentin and β-actin. Up-regulated proteinsincluded the antioxidants superoxide dismutase, thioredoxin, andcytochrome c reductase. Oxidative stress can also lead to dysfunction ofthe proteasome and is implicated in PD pathogenesis (Gu et al. (2005)Cell Death Differ., 12:1202-1204). Indeed, as a result of N-α-synstimulation the proteasome 26S subunit was decreased in these celllysates, although ubiquitin and the ubiquitin conjugating enzyme E2Nwere increased, suggesting that the microglia may be compensating fordecreased proteasomal activity (FIG. 27).

Neuroinflammatory Parkinson's Disease Phenotype

Analysis of the proteome of N-a-syn-stimulated microglia revealed theinduction of NF-κB-related signaling pathways and initiation of severalproteins involved in the cellular response to inflammation and oxidativestress. To investigate whether differential expression of proteinsidentified in the proteomic analyses of in vitro stimulated microgliawas reflected in PD, protein expression of lysates prepared from the SNand BG of control and PD brains were assessed by western blot assays(FIG. 29). Proteins increased in abundance within the secretome as aresult of N-α-syn stimulation were cross-validated in PD patientsincluding calmodulin and the redox-associated secreted proteinsbiliverdin reductase and thioredoxin; whereas, secretion of theregulatory proteins glatectin-3 and 14-3-3σ, structural protein actin,and the redox protein glutathione-S-transferase were decreased followingN-α-syn stimulation. These analyses verified the increased expression ofcalmodulin as well as the antioxidant biliverdin reductase in the SN ofPD compared with age-matched controls without neurological disease.Actin expression appeared decreased in PD brains relative to controls,which coincided with the analysis of the N-α-syn-stimulated microgliasecretome. In contrast to the in vitro results, expression of 14-3-3σand galectin 3 were increased in PD brains. Glutathione-S-transferaseexpression was decreased in PD brains relative to control. Althoughexpression of thioredoxin did not appear to be different within the SN,expression in the BG was significantly decreased in PD. Proteins thatwere identified in the proteome of N-α-syn-stimulated microglia were, inpart, also cross-validated in SN of PD and control brains. Akin to thelaboratory model, expression of calmodulin was increased whereasexpression of α-enolase, L-plastin, α-tubulin, and actin were decreasedin PD relative to control. The discrepancies between the cellular modeland expression in the human tissue underscore the complexity of humandisease and the multiple cell components that are involved. Indeed,comparing non-affected brains to PD brains may be misleading as alreadythe proportion of cellular components are different, especially at endstage where greater than 80% of the dopaminergic neurons have died andsubstantial gliosis is present. However, overall these results supportthat the molecular and biochemical analyses of N-α-syn microglialactivation appear, in part, applicable to human PD.

Example 5

Activated microglia are linked to Parkinson's disease (PD) pathobiology(McGeer et al. (1988) Neurology 38:1285-1291; Hald et al. (2007)Subcell. Biochem., 42:249-279; Whitton, P. S. (2007) Br. J. Pharmacol.,150:963-976; Wilms et al. (2007) Curr. Pharm. Des., 13:1925-1928; Yuanet al. (2007) Neurosci. Bull., 23:125-130). The primary mediators ofneuroinflammatory responses in PD are activated microglia. How suchmicroglial activation can be regulated could present diagnostic andtherapeutic options for PD (Hermanowicz, N. (2007) Semin. Neurol.,27:97-105; Klegeris et al. (2007) Curr. Opin. Neurol., 20:351-357;Lipton et al. (2007) Int. Rev. Neurobiol., 82:1-27; Reynolds et al.(2007) Int. Rev. Neurobiol., 82:297-325). This is of importance as PDremains the second most common neurodegenerative disorder among theelderly and will increase in incidence and prevalence as the baby boomergeneration ages (Khandhar et al. (2007) Dis. Mon., 53:200-205). PD ischaracterized by progressive loss of dopaminergic neurons in thesubstantia nigra pars compacta (SNpc) and their projections to thecaudate-putamen of the basal ganglia (BG). A pathological hallmark ofdisease is the presence of fibrillar α-synuclein (α-syn) inclusionsknown as Lewy bodies (LB) in the SN that are associated withdegenerating neurons (Hodaie et al. (2007) Neurosurgery 60:17-28.28-30). Although the etiology of PD remains unknown, a large body ofevidence links inflammation, mitochondrial dysfunction, oxidativestress, and diminished neurotrophic support to disease (Przedborski, S.(2005) Parkinsonism Relat. Disord., 11(Suppl 1):S3-7; Zhang et al.(2005) Faseb J., 19:533-542; Mandemakers et al. (2007) J. Cell Sci.,120:1707-1716).

Activated microglia are closely associated with dying or damageddopaminergic (DA) neurons (McGeer et al. (1988) Neurology 38:1285-1291;Czlonkowska et al. (1996) Neurodegeneration 5:137-143). A link betweenmicroglial secretory activities and neurodegeneration is made throughthe plethora of neurotoxic factors they produce following activationincluding tumor necrosis factor alpha (TNF-α, reactive oxygen species(ROS), interferons, excitatory amino acids, interleukin (IL)-1β, IL-6,nitric oxide (NO), and leukotrienes (Rogove et al. (1998) Curr. Biol.,8:19-25; Wu et al. (2002) J. Neurosci., 22:1763-1771). There iscompelling evidence that the release of aggregated and nitrated α-syn(N-α-syn) from dying or damaged SN dopaminergic neurons serves, in part,to provoke a neuroinflammatory response (Zhang et al. (2005) Faseb J.,19:533-542; Thomas et al. (2007) J. Neurochem., 100:503-519) that, leftuncontrolled, contributes to neurodegenerative activities and the tempoof disease.

The mechanism of microglia-mediated DA neurotoxicity is linked to thegeneration of oxidative insult from microglia. DA neurons, inparticular, possess reduced antioxidant capacity as a result of lowintracellular glutathione, which renders DA neurons vulnerable tooxidative stress relative to other cell types (Loeffler et al. (1994)Clin. Neuropharmacol., 17:370-379). Ongoing investigations haveidentified cellular properties including ROS production, morphologicaltransformation, inflammatory cytokine secretion, nuclear factor-kappa B(NF-κB) activation, and a proteome characteristic of an inflammatoryresponse that accompany microglial stimulation with N-α-syn and DAdegeneration. Nonetheless, PD progresses slowly over the span of severalyears to decades suggesting that in addition to neuroinflammation, acompensatory regulatory mechanism is operative for disease (Przedborski,S. (2005) Parkinsonism Relat. Disord., 11(Suppl 1):S3-7).

Activated microglia possess dual roles for neural repair and diseasethat are dependent upon specific environmental cues, degree of injury,stage of disease, and brain regions involved. In previous works, it hasbeen demonstrated that stimulation of microglia with sciatic and opticnerve fragments, in addition to inducing expression of pro-inflammatorycytokines, incites a neuroprotective phenotype by upregulation ofseveral signal transducer and activator of transcription (STAT) genes,cytoskeletal proteins, lysosomal proteins, and immunoregulatoryproteins, and enhanced expression of neurotrophins including brainderived neurotrophic factor (BDNF) and glial derived neurotrophic factor(GDNF) (Glanzer et al. (2007) J. Neurochem., 102:627-645). Based onthose observations, it was reasoned that microglial response to N-α-synmight also induce underlying compensatory or protective mechanisms tocircumvent the injurious effects. Herein, a dual profile for both atoxic and trophic microglial cell in response to N-α-syn isdemonstrated. This profile indicates modulation of theglutamate-glutamine cycle and an upregulation of cytoskeletal proteins,regulatory and redox-active proteins. Most importantly, high levels ofcysteine were produced in parallel to reduced cathepsin and highcystatin levels. It is demonstrated herein that microglia acquire aneurotoxic phenotype after aggregated N-α-syn stimulation. However, atandem compensatory response through regulation of cysteine secretion,cystatin expression, cathepsin activity, and NF-κB activation wasobserved. These data provide a yet undefined regulatory role formicroglia in PD pathobiology.

Materials and Methods Purification, Nitration, and Aggregation ofRecombinant Mouse α-Syn

Purification, nitration, and aggregation of recombinant mouse α-syn wereperformed as previously described. Protein concentration was determinedfrom the dry weight of the lyophilized protein.

Isolation, Cultivation, and N-α-syn Activation of Murine Microglia

Microglia from C57BL/6J mice neonates (1-2 days old) were prepared usingpreviously described techniques (Dobrenis, K. (1998) Methods16:320-344). All animal procedures were in accordance with NationalInstitutes of Health guidelines and were approved by the InstitutionalAnimal Care and Use Committee of the University of Nebraska MedicalCenter. Brains were removed and placed in Hanks' balanced salt solution(HBSS) at 4° C. The mixed glial cells were cultured for 7 days incomplete Dulbecco's modified Eagle's medium (DMEM) containing 10% FBS(fetal bovine serum), 10 μg/ml gentamicin and 2 μg/ml macrophage colonystimulating factor (MCSF; Wyeth, Inc., Cambridge, Mass.). To obtainhomogenous microglial cell populations, culture flasks were gentlyshaken and nonadherent microglia were transferred to new flasks. Theflasks were incubated for 30 minutes to allow the microglia to adhere,and loose cells removed by washing with DMEM. Microglia were plated at2×10⁶ cells/well in 6-well plates in complete DMEM. The adherentmicroglia obtained using this method was assessed for purity byimmunocytochemical analysis for CD11b positive cells and bymorphological examination. As previously reported, the microglial cellpopulation was >98% CD11b+ (Enose et al. (2005) Glia 51:161-172). Oneweek following re-plating, cells were stimulated with 100 nM ofaggregated N-α-syn/well or no stimulation for 4 hours and 24 hours.Media were replaced with serum free DMEM without phenol red or otheradditives (Invitrogen/GIBCO) and incubated for 24 hours in a 37° C., 5%CO₂ incubator.

Surface Enhanced Laser Desorption Ionization-Time of Flight (SELDI-TOF)

Protein profiling of culture supernatants was performed using SELDI-TOFProteinChip® assays (Enose et al. (2005) Glia 51:161-172; Kadiu et al.(2007) J. Immunol., 178:6404-6415) (Ciphergen Biosystems, Fremont,Calif.). The normal phase NP20 protein chip was selected to profileculture supernatants for low and high abundant proteins based on thenon-discriminating binding affinity of its hydrophobic surface toproteins regardless of their chemical structure. An aliquot of culturesupernatant (2 μg of protein) was applied onto each spot and air-dried.To each air-dried spot, 0.5 μl of 50% sinapic acid (SPA) was added andair-dried. The 50% SPA was prepared as a saturated solution in solventcontaining 30% acetonitrile (ACN), 15% isopropanol, 0.5% trifluoroaceticacid, and 0.05% Triton X-100. SELDI-TOF spectra were generated onsupernatants collected from three separate microglia cultures.Supernatants collected at each time point were run in triplicate, onthree protein chips at different spots to control for instrumental andexperimental variability. Molecular mass/charge (m/z) ratios of laserbeam ionized proteins were measured in a ProteinChip® PBS II reader. TheProteinChip® Reader was externally calibrated for each analysis usingthe four standard proteins: bovine insulin (5,733.6 Da), cytochrome C(12,230.9 Da), superoxide dismutase (SOD) (15,591.4 Da), andbeta-lactoglobulin (18,363.3 Da). Acquired spectra were analyzed usingProteinChip® software 3.2 (Ciphergen Biosystems). The ProteinChip®&analyses were performed in three independent experiments, and the dataset from each microglia comprised a minimum of 8 spectra. All spectrawere combined in one file and normalized to total ion current. Peakswere automatically detected using the Biomarker Wizard of ProteinChip®software 3.2. Peak detection parameters were first pass signal/noise(S/N) ratio=5, second pass S/N ratio=2, mass tolerance=0.5%, andestimated peaks were included in completion of clustering.

Protein identification by LC-MS/MS

Following in gel tryptic digestion and column purification, driedpeptide samples from cell supernatant fluids were reconstituted in 0.1%formic acid/HPLC-grade water, detected on a ProteomeX LCQ™ DECA XP Plusmass spectrometer (ThermoElectron, Inc. Waltham, Mass.), and identifiedusing BioWorks 3.1 SR software. Proteins identified by peptides having aUnified Score (BioWorks 3.1 SR, ThermoElectron, Inc. Waltham,Mass.) >3000 were marked for further analysis (Enose et al. (2005) Glia51:161-172; Kadiu et al. (2007) J. Immunol., 178:6404-6415).

Extracellular Supernatant Fractionation

Culture supernatants were concentrated using Centriplus™ centrifugalfilter devices (Millipore, Billerica, Mass.) and dialyzed against MiliQwater using Cellu-Sep® H1 cellulose membranes (Membrane FiltrationProducts). Samples of culture supernatants were fractionated using 1DSDS-PAGE. Each 200 μg sample was diluted with NuPAGE® loading buffer andseparated using NuPAGE® Novex 10% Bis-Tris (Invitrogen) gel. Afterelectrophoresis, the gels were stained with Coomassie Brilliant blueG-colloidal concentrate (Sigma-Aldrich, St. Louis, Mo.).

Nuclear/Cytosol Fractionation

Cell lysates were prepared from N-α-syn-stimulated and controlmicroglia. Cells were rinsed 3× with PBS, following gentle scraping,cells were collected following centrifugation at 600×g for 5 minutes.Cytosol and nuclear fractions were prepared using the Nuclear/CytosolFractionation Kit (BioVision; Mountain View, Calif.) according tomanufacturer's instructions.

Western Blot Assays

A total of 20 μg of each sample was loaded onto 4-12% Bis-Tris NuPAGENovex gels (Invitrogen) and transferred onto PVDF membranes (BioRad,Hercules, Calif.). Primary antibodies to calmodulin (1:1000) and 14-3-3σ(1:200) (Millipore), biliverdin reductase (1:5000), thioredoxin(1:2000), β-actin (1:5000), ferritin light:chain (1:1000), galectin 3(1:1 000) (Abcam, Cambridge, Mass.), NF-κB p65 and p50 (1:200) (SantaCruz Biotechnology, Inc., Santa Cruz, Calif.), cystatin B (1:500), andcathepsin B (1:1000) (R & D Systems) were used for analyses. Blots wereprobed with the respective horseradish peroxidase-conjugated secondaryantibodies (1:5000; Invitrogen) and detected using SuperSignal® WestPico Chemiluminescent substrate (Pierce Biotechnology, Inc). Theintensity of protein bands was quantified using ImageJ and normalized toGapdh (1:5000, Santa Cruz Biotechnology, Inc.).

Metabolite Assays

Microglia were cultured with and without N-α-syn in media without addedglutamine for 2, 4, 8, and 24 hours during stimulation. For analysis ofextracellular metabolites, culture supernatants were collected at eachtime point and mixed with equal volumes of metaphosphoric acid solution(16.8 mg/ml HPO₃, 2 mg/ml EDTA and 9 mg/ml NaCl). For analysis ofintracellular metabolites, cells were washed three times with ice coldPBS, maintained on ice in PBS, and detached with a tissue culturescraper. To measure protein concentration, an aliquot of the cellsuspension was mixed with an equal volume of lysis buffer (0.1 M sodiumphosphate, pH 7.4, containing 0.1% Triton-X100, 10 μl/ml proteaseinhibitor cocktail (Sigma), 25 μg/ml tosyllysine chloromethylketone and5 μg/ml phenylmethylsulfonyl fluoride). Samples were stored at −80° C.until further use. Data are representative of three independentexperiments performed in triplicate.

High Performance Liquid Chromatography (HPLC) Analyses

For analysis of metabolites, the metaphosphoric acid fixed samples ofcells or culture supernatant were thawed, vortexed, and clarified bycentrifugation at 14000×g for 10 minutes at 4° C. Thiol metabolites inprotein free extracts were derivatized with monoiodoacetic acid (7 mM)followed by mixing with an equal volume of 2,4-dinitrofluorobenzenesolution (1.5% v/v in absolute ethanol) and analyzed by HPLC usingu-Bondapak-NH2 300×3.9 mm column (Waters) with a methanol-acetategradient as described previously (Mosharov et al. (2000) Biochemistry39:13005-13011). The concentration of metabolites in the samples wasdetermined using a standard curve generated for each metabolite ofinterest. Results were normalized to protein concentration in eachsample. The protein concentration in samples was measured using theBradford reagent (Bio-Rad; Hercules, Calif.) with bovine serum albuminas standard.

Intracellular Glutathione (GSH)

Microglia were cultured with and without N-α-syn for 24 hours in mediawithout added glutamine. Cells were washed three times with ice coldPBS, and detached with a tissue culture scraper. Cell suspensions werecollected in triplicate and assayed for GSH, GSSG, and total glutathionelevels with the Biovision Glutathione Assay Kit (Biovision, MountainView, Calif.) according to manufacturer's protocol. Briefly, cells werehomogenized in 100 μl assay buffer, and glutathione was stabilized with6N perchloric acid. For assay, a standard curve was performed with GSHstandard. Samples were prepared according to protocol for determinationof GSH, GSSG, and total glutathione. Reducing Agent Mix (Biovision) wasadded to convert GSSG to GSH. An o-phthalaldehyde probe was added to thesamples for 40 minutes. Samples were then assessed in a 96 wellfluorometer plate using a SpectraMAX GEMINI (Molecular Devices,Sunnyvale, Calif.) at excitation/emission of 340/450 nm.

Cathepsin B Activity

Microglia were seeded onto sterile glass coverslips at 10⁵ cells perwell and allowed to adhere for 24 hours prior to stimulation withaggregated N-α-syn. Cathepsin B activity was determined using theCV-Cathepsin B Detection Kit (BIOMOL International LP). For measurementof cathepsin B activity, arginine conjugated cresyl violet [CV-(RR)₂, ared fluorogenic substrate when unconjugated] was added to the culturemedia and allowed to incubate for 60 minutes in a 37° C., 5% CO₂incubator. The attached ArgArg group is a substrate for cathepsin Bcleavage. Hoechst stain was added at 0.5% v/v and incubated with cellsfor an additional 5 minutes to stain nuclei. Fluorescence intensity ofunconjugated CV was determined at 550 nm for excitation and 610 nm foremission of 3 wells/10 fields per well/experimental group. MeanFluorescence intensity of unconjugated CV was normalized to the meanintensities of Hoechst staining at 480 nm for excitation and 540 nm foremission for each sample.

Cathepsin B Inhibition and Neurotoxicity

MES 23.5 cells were cultured in 75-cm² flasks in DMEM/F12 with 15 mMHEPES (Invitrogen) containing N2 supplement (Invitrogen), 100 U/ml ofpenicillin, 100 μg/ml streptomycin, and 5% FBS. MES23.5 cells is a cellline derived using somatic cell fusion of rat embryonic mesencephaloncells and the murine neuroblastoma-glioma cell line N18TG2 that producedopamine and express tyrosine hydroxylase (Crawford et al. (1992) J.Neuroscience 12:3392-3398). Cells were grown to 80% confluence thenplated at a density of 10×10⁴ cells on sterile glass coverslips.Microglia were plated in 6 well plates and incubated for 1 hour with orwithout 1 μM of cathepsin B inhibitors, CA-074 [cell impermeable] orCA-074 Me [cell permeable] (Sigma), in a 37° C., 5% CO₂ incubator toinhibit residual cathepsin B expression (Gan et al. (2004) J. Biol.Chem., 279:5565-5572). N-α-syn (100 nM) was then added directly to thismedia for 24 hours. Unstimulated microglia and N-α-syn stimulation aloneserved as controls. Following stimulation, supernatants were removed andadded to cultures of MES23.5 cells for an additional 24 hours. MES 23.5cell viability was assessed using the Live/Dead cytotoxicity assay(Invitrogen) as previously described. Live cells were distinguished bythe uptake of calcein AM to acquire a green fluorescence[excitation/emission (ex/em) 495/515=m], while dead cells acquired a redfluorescence (ex/em 495/635 μm) due to the uptake of ethidiumhomodimer-1 (EthD-1). Cell enumerations were performed usingfluorescence microscopy (200× magnification, n=6 wells per group, 3frames per well).

Statistical Analyses

All values are expressed as means±SEM. Differences among means wereanalyzed by oneway ANOVA followed by Bonferroni post-hoc testing forpair-wise comparison.

Results

N-α-syn-Activated Microglia Secretions and Neuroinflammatory Responses

In previous studies it has been demonstrated that a temporal pattern ofmicroglial activation occurs following stimulation of microglia withN-α-syn, and consists of increased expression of factors attributed toinflammatory responses, oxidative stress and significantneurocytotoxicity (Zhang et al. (2005) Faseb J., 19:533-542). Thesecretion of potentially neurotoxic compounds characterizes theprogression of an activated microglial phenotype to a neurotoxicphenotype, while secretion of trophic factors that support neuronalsurvival and cell-cell communication characterizes a quiescentmicroglial phenotype. To analyze the secretory profile induced uponN-α-syn stimulation, supernatants from microglia stimulated for 4 hourswith N-α-syn were collected at 8, 16, and 24 hours post-stimulation.Supernatants were first screened for low and high abundant proteinsusing SELDI-TOF analysis. In these experiments, spectra were generatedfrom cell supernatants collected from control and stimulated cultures ateach time-point in three separate experiments using a NP20 Protein Chip.Signal to noise ratios of 5 and 2 for first and second passesrespectively and 0.5% mass tolerance (Enose et al. (2005) Glia51:161-172). Representative spectra of culture supernatants obtainedfrom microglia simulated with aggregated N-α-syn revealed thatmicroglial secretory constituents were significantly altered compared tothose of unstimulated microglial controls (FIG. 30A), and consisted ofseveral peaks coinciding with molecular masses of calcium regulatoryproteins (calcyclin, 10,051; calmodulin, 16,706 Da; calvasculin, 11,721Da), redox-active proteins (thioredoxin, 11,544 Da), and TNF-α (17, 907Da). In contrast, secretory profiles of microglia cultured in thepresence of unaggregated N-α-syn revealed similar profiles tounstimulated control microglia. These results determined that microglialsecretory products were significantly changed by stimulation withN-α-syn, and encouraged the pursuit of 1D SDS PAGE methods to isolateand identify differentially secreted proteins upon stimulation. Use of1D SDS PAGE followed by LC-MS/MS analysis has been shown to be reliablein identifying differentially expressed proteins (Enose et al. (2005)Glia 51:161-172; Glanzer et al. (2007) J. Neurochem., 102:627-645;Ciborowski et al. (2007) Virology 363:198-209) and identification ofN-α-syn following immunopreciptiation with confirmatory western blotanalyses. LCMS/MS analyses of SDS-PAGE fractions identified 30 proteinscommon to supernatants from both stimulated and control microglia.Analysis of proteins secreted from N-α-syn-stimulated microglia byLC-MS/MS identified those proteins detected by SELDI-TOF. Microglialexpressed proteins were considered using criteria that at least twopeptides from a protein were detected with a unified score (BioWorks,3.1 SR) greater than 3000 as previously described (Glanzer et al. (2007)J. Neurochem., 102:627-645). Proteins were identified as either greateror lower in abundance based on the number of peptides detected byLC-MS/MS, or the presence of a protein identified with high confidencein culture supernatants of one group but not in culture supernatants ofthe other. Altogether, LC-MS/MS analysis revealed 40 increased and 34decreased in abundance within culture supernatants when compared tounstimulated controls (FIGS. 30D and 30E); seven of which were validatedby western blot analysis (FIG. 30C) including decreased secretion ofactin, galectin 3, and 14-3-3 sigma along with increased expression ofbiliverdin reductase, calmodulin, ferritin light chain, and thioredoxinin culture supernatants of N-α-syn-stimulated microglia. Sixteenproteins found in greater abundance in supernatants of aggregatedN-α-synstimulated microglia were classified as being involved in bothcellular activation and regulation. For example, regulators of oxidativestress including thioredoxin, biliverdin reductase, and ferritin lightchain were abundantly secreted by stimulated microglia. Also found inthese supernatants were cell-morphogenesis proteins L-plastin,actin-related protein 3 homolog B (ARP3) and adenylyl cyclase-associatedprotein 1 (CAP 1); the lysosomal proteins, α-N-acetylglucosaminidase(NAGLU) and N-acetylgalactosamine-6-sulfate sulfatase (GALNS); and thecalcium binding proteins EF-hand domain-containing protein 2 (EFHD2,swiprosin1) and nucleobindin (Islam et al. (2006) J. Biol. Chem.,281:6860-6873). Proteins less abundant in supernatant fluids fromN-α-syn-stimulated microglial supernatants including calcyclin, β-actin,histone H4, triose-phosphate isomerase, phosphoglycerate mutase 1,cathepsin S, 14-3-3σ, and ubiquitin were reported to be associated withexosomal vesicles, whereas only three of the proteins (ferritin lightchain, CAP1, and L-plastin) that were more abundant inN-α-syn-stimulated microglial supernatants have been associated withexosomes (Thery et al. (2001) J. Immunol., 166:7309-7318; Wubbolts etal. (2003) J. Biol. Chem., 278:10963-10972; Pisitkun et al. (2004) Proc.Natl. Acad. Sci., 101:13368-13373; Potolicchio et al. (2005) J.Immunol., 175:2237-2243; Faure et al. (2006) Mol. Cell. Neurosci.,31:642-648).

Metabolic Response of Microglia to N-α-Syn Stimulation

In addition to activation of signaling pathways that inciteneuroinflammatory responses, increased expression and secretion ofredox-active proteins suggested that a multifaceted microglial responseto N-α-syn may affect PD, and that regulatory mechanisms which counteroxidative stress may accompany the inflammatory response. To assess thelatter possibility, the concentrations of metabolites involved in theglutamate-glutamine cycle were determined. It was theorized that as aresult of stimulation with N-α-syn, changes in the levels of microglialmetabolites would occur relative to unstimulated controls (FIG. 31A).Stimulation with N-α-syn resulted in increased secretion of glutamate insupernatants collected at time points measured over a 24 hour timeperiod compared to supernatants collected from unstimulated controlcells (32.5±0.5 μM versus 21.0±1.0 μM, P<0.0001 at 8 hours ofstimulation) and continued to rise, so that by 24 hours theconcentration was 45.5±1.5 μM compared to 26.5±2.5 μM for control(P<0.0001). Moreover, extracellular cysteine was also foundsignificantly elevated in response to N-α-syn compared to controls(16.6±0.7 μM versus 6.96±0.03 μM, P<0.0001; 8 hours). As expected,increased extracellular cysteine was accompanied by decreased cystine.This was seen at 24 hours following exposure to N-α-syn (270.0 μM versus286.0±3.0 μM in controls, P<0.0001 at 24 hours). Intracellularconcentrations of both glutamate and cysteine were decreased instimulated microglia compared to controls (FIG. 31B).

Determination of intracellular GSH concentration by HPLC indicated thatexposure to N-α-syn resulted in its 2 fold rapid depletion at 2 hours to31.3±0.6 μmoles/g protein compared to controls with 60.0±3.3 μmoles/gprotein (data not shown, P<0.001). During continuous exposure withN-α-syn, glutathione levels rose to nearly pre-stimulatory levels by 8hours (69.0±2 μmoles/g protein versus 111±2 μmoles/g protein), and thelevels again dropped by 24 hours (49±1 μmoles/g protein). In contrast,glutathione levels steadily rose in cell lysates of unstimulatedmicroglia to 126±1 μmoles/g protein (P<0.0001, FIG. 2B) by 24 hours. Inaddition, the ratio of GSH to oxidized glutathione (GSSG), an indicatorof cellular redox status, declined steeply within 2 hours in response toN-α-syn stimulation (34.2±1.0 versus 89.4±2.6, P<0.001), however thisratio gradually rebounded (70.0±2.0 and 65.0±1.0 by 8 and 24 hoursrespectively) but remained at about 2 fold lower throughout the courseof stimulation in comparison to unstimulated control (114.0±3.0 and163.0±1.0 by 8 and 24 hours respectively). Investigation of GSH and GSSGlevels using the Biovision glutathione assay kit confirmed theseresults, and revealed that by 24 hours the GSH levels were significantlydecreased (P<0.001), and corresponded with decreased GSH/GSSG ratio andtotal GSH plus GSSG levels compared to levels in unstimulated celllysates.

Cathepsin B Activity and N-α-Syn Microglial Activation and Cytotoxicity

Microglia have been shown to express a number of proteases, includingthe cysteine protease cathepsin B, where it may play a role indegradation of matrix proteins and associated signal transductionmolecules that can induce neuronal apoptosis (Kingham and Pocock, 2001).To further investigate the microglial response to N-α-syn in regulatingneurotoxicity a series of tests were performed to assess thedifferential expression of cathepsin B, and the cysteine proteaseinhibitor cystatin B, over the course of stimulation. Western blotanalysis revealed a significant increase in expression of cystatin B inmicroglial cell lysates following 4 hours of N-α-syn stimulationcompared to unstimulated controls and decreased expression of cathepsinB (FIG. 32A). However, by 24 hours of N-α-syn stimulation, cystatin Bexpression was decreased, whereas expression of cathepsin B in celllysates was not significantly changed. Since regulation of cathespin Bis primarily post-translational, increased expression of cystatin Bwould more closely correspond to decreased cathepsin B activity ratherthan decreased protein expression, therefore it was investigated theenzymatic activity of cathespin B before and during stimulation withN-α-syn. Activity of intracellular cathepsin B was significantlydecreased in microglia following 4 hours of stimulation with N-α-syncompared to basal activity before stimulation, and then enzymaticactivity increased significantly over pre-stimulatory level by 24 hours(FIG. 32B). It was next investigated whether cathepsin B could alsocontribute to N-α-syn stimulated microglial cytotoxicity. Microglia werestimulated with N-α-syn in the presence of either the selectivecathepsin B inhibitor CA-074 or the cell permeable form CA-074 Me for 24hours, when cathepsin B activity was elevated following N-α-synstimulation alone. The resultant supernatants were then added tocultures of dopaminergic MES23.5 cells, for an additional 24 hours forcell death measurement (FIG. 33). Supernatants from unstimulatedmicroglia did not induce DA cell death, while N-α-syn stimulation aloneresulted in significant cytotoxicity (26% of control, P<0.001).Inhibition of secreted Cathepsin B by the cell impermeable inhibitor,CA-074, partially protected DA cells against N-α-syn microglial mediatedcell death (53% protection v. N-α-syn alone, P<0.01), while the cellpermeable form, CA-074 Me resulted in heightened DA cell protection(87%, P<0.001).

NF-κB Subunit Translocation and N-α-syn Microglial Activation

Activation of the NF-κB pathway leads to the production of inflammatorymediators implicated in inducing neuronal injury (Qin et al. (2005)Blood 106:3114-3122). Indeed, upregulation of NF-κB transcription isinduced upon stimulation with N-α-syn activation, as well as beingsignificantly increased in the SN of post-mortem brains from PDpatients. In addition, analysis of the microglial secretome identifiedmany proteins, including for example phosphatidylethanolamine-bindingprotein, thioredoxin, and FK506-binding protein 12 (FKBP1A) that aredownstream of either the NF-κB or mitogen-signaling pathways, suggestingthe involvement of NF-κB pathway to initiate not only the inflammatoryresponse, but also regulation of the response. To investigate whetherthis dual toxic and trophic reaction to N-α-syn by microglia wascontingent on activation of the NF-κB pathway, NF-κB translocation tothe nucleus was assessed where it can bind DNA and activatetranscription of genes encoding proteins involved in an inflammatoryresponse. As early as 1-1.5 hours stimulation of microglia with N-α-syn,NF-κB p50 and p65 were increased in the nucleus, and coincided withdecreased expression of these subunits within the cytosolic fractions(FIG. 34A). By 4 hours stimulation however, the nuclear localization ofNF-κB subunit p50 was decreased compared to 1.5 hours stimulation, andremained repressed through 8 hour. By 24 hours of stimulation, the p50subunit was once again diminished in the cytosol and increased withinthe nucleus (FIG. 34B). Notably, compared to 0 hour, p65 levels in thenucleus were high with correspondingly diminished levels within thecytosolic fraction by 1 hour stimulation (FIG. 34C). These data suggestthat the microglial compensatory response, albeit temporary, is alsoreflective of discontinuous activation of the NF-κB pathway anddownstream inflammatory cascades.

Example 6

An inciting event that underlies Parkinson's disease (PD) neurobiologyis the accumulation of aggregated proteins within neuronal cell bodiesand microglia is associated with microglial activation and neuronaldeath. Deposition of misfolded and nitrated alpha-synuclein (N-α-syn)into Lewy bodies (LB) within nigral dopaminergic neurons of thesubstantia nigra pars compacta (SNpc) (Spillantini et al. (1997) Nature388:839-840; Giasson et al. (2000) Science 290:985-989), with subsequentrelease into extracellular spaces and draining cervical lymph nodesaffect neuronal loss by engaging innate and adaptive immune responses(Lee et al. (2008) Biochem. Biophys. Res. Commun., 372:423-428; Theodoreet al. (2008) J. Neuropathol. Exp. Neurol., 67:1149-1158). This leads tooxidative stress, microglial and APC activation, and neuronaldegeneration (Thomas et al. (2007) J. Neurochem., 100:503-519).Interestingly, α-syn immunization was shown to generate humoralresponses for clearing protein aggregates (Masliah et al. (2005) Neuron46:857-868). However, using nitrated forms of α-syn (N-4YSyn) as animmunogen profound effector T cell (Teff) responses were shown toexacerbate neuroinflammation, and neurodegeneration analogous to theuntoward T cell-mediated meningoencephalitic responses observed by Aβimmunization (Smith et al. (2002) Lancet 359:1864-1865). Others reportedthat T cell responses elicited during the course of1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) intoxication led toaccelerated neurodegeneration (Theodore et al. (2008) J. Neuropathol.Exp. Neurol., 67:1149-115).

Interestingly, components of adaptive immunity affect neural repair andprotection. Indeed, regulatory T cells (Treg) protect againstMPTP-induced dopaminergic degeneration. This raised the specter ofopposing effects for CD4+ T cell subsets on brain disease whereauto-aggressive Teff responses speed the tempo of disease Treg attenuateneurodegeneration. This is in keeping with known anti-inflammatory andneurotrophic function of Treg, their essential role in controllingimmune-mediated inflammation and mononuclear phagocyte phenotype(Cederbom et al. (2000) Eur. J. Immunol., 30:1538-1543; Thornton et al.(2000) J. Immunol., 164:183-190; Tiemessen et al. (2007) Proc. Natl.Acad. Sci., 104:19446-19451). Thus, it was investigated whether anadjuvant to promote specific adaptive immune responses could be usedwith N-α-syn as a vaccine for PD. Using vasoactive intestinal peptide(VIP), a neuropeptide known to induce Treg responses (Delgado et al.(2005) J. Leukoc. Biol., 78:1327-1338; Gonzalez-Rey et al. (2006) Blood107:3632-3638), it is now show that replacement of functional Tregwithin the N-α-syn splenocyte mixture results in neuroprotection and ismodulated through altered Th17 expression.

Materials and Methods Animals, Immunizations, and MPTP Intoxication

Recombinant C-terminal tail of α-syn (4YSyn) was purified, nitrated(N-4YSyn), and tested for endotoxin as described. Male C57BL/6J mice andFoxP3-GFP knock in C57BL/6J mice (5 weeks old, The Jackson Laboratory)were immunized with N-4YSyn emulsified in adjuvant as described. Donormice that did not receive immunizations were injected i.p. with 15 μg ofVIP (Sigma-Aldrich) in PBS. Recipient mice received four i.p. injectionsat 2 hour intervals of either vehicle (PBS, 10 mL/kg bodyweight) orMPTP-HCl (16 mg MPTP/kg bodyweight of free base in PBS; Sigma-Aldrich).Twelve hours after the last injection, MPTP-intoxicated mice receivedadoptive transfers of whole SPC, Treg or no cells (n=5-7 mice per groupper time point). On days 2 and 7 post-MPTP, mice were sacrificed andbrains were processed for analysis. All animal procedures were inaccordance with National Institutes of Health guidelines and wereapproved by the Institutional Animal Care and Use Committee of theUniversity of Nebraska Medical Center. MPTP safety measures were inaccordance with published guidelines (Przedborski et al. (2001) J.Neurochem., 76:1265-1274).

Isolation and Adoptive Transfer of SPC and CD4+ CD25+T Cells.

Seven days following treatment, mice were sacrificed and single cellsuspensions were prepared from inguinal lymph nodes and spleens. CD4+ Tcell populations from spleens and lymph nodes were enriched by negativeselection with CD4-enrichment columns (R & D Systems) followed byCD25-PE positive selection with AutoMACS (Miltenyi Biotec). T cells werecultured in RPMI medium 1640 (Invitrogen) supplemented with 10% FBS, 2mM L-glutamine, 25 mM HEPES, 1 mM sodium pyruvate, 1× nonessential aminoacids, 55 μM 2-mercaptoethanol, 100 units/ml penicillin, 100 μg/mlstreptomycin (Mediatech) in the presence of anti-CD3 (145-2C11; BDPharmingen), 4YSyn or N-4YSyn. Proliferation and inhibition assays wereperformed as described. MPTP-intoxicated mice received an i.v. tailinjection of 5×10⁷ SPC or 1×10⁶ Treg in 0.25 ml of HBSS.

In Vitro Polarization of CD4+ T Cells

CD4+ T cells were isolated from N-4YSyn immunized donors and cultured1×10⁶/ml CD4+ T cells with 2×10⁶/ml irradiated SPC and 10 mg/ml N-4YSynin 20 ml of complete RPMI 1640 supplemented with 10% FBS, 2 mML-glutamine, 10 mM HEPES, 55 μm 2-mercaptoethanol, 100 units/mlpenicillin, and 100 μg/ml streptomycin in T25 flasks. For polarizationthe CD4+ T cells were cultured with 10 ng/ml IL-2 for Th0; 10 ng/mlIL-12 and 2 μg/ml anti IL-4 for Th1; 10 ng/ml IL-4 and 2 μg/mlanti-IL-12 for Th2; and 3 ng/ml TGF-0, 10 ng/ml IL-6, 5 ng/ml IL-1b, 10ng/ml IL-23, 2 μg/ml anti-IL-4, 2 μg/ml anti-IL-12, 2 μg/ml anti-IFN-g,and 2 μg/ml anti-IL-2 (Laurence et al., 2007 Immunity 26:371) for 5days. The Th subsets were then harvested, and 10×10⁶ T cells transferredto each recipient. For stimulation of cytokine production, Th subsetswere stimulated with 20 ng/ml PMA and 1 μM ionomycin (Sigma-Aldrich) for5 hours and supernatants collected 24 hours later for analysis.

Flow Cytometric Analysis

Samples from cell fractions were labeled with fluorescently labeledantibodies (eBiosciences) and analyzed by flow cytometry with aFACSCalibur flow cytometer (BD Biosciences).

RNA Isolation and Real-Time PCR Arrays

RNA was purified using TRIzol reagent (Invitrogen Corp) and the RNeasyMini Kit (QIAGEN Sciences), prior to cDNA synthesis. Real-time PCRanalysis using pathway-focused gene expression profiling arrays (SABiosciences) was performed according to manufacturer's protocol.

Cytokine Analyses

A multi-analyte cytokine ELISArray (SA Biosciences) was used forcytokine analysis within cell culture supernatants according tomanufacturer's protocol, Absorbance values were read at 450 nm afterstopping the reaction. A cytometric bead array for Th1/Th2 cytokines andan IL-17a Flex set were used to quantitate cytokine concentrationswithin culture supe matants. The bead arrays were performed according tomaunfaturer's protocol and the data acquired on a BD FACSArraybioanalyzer and analyzed using the FCAP Array Software (BD biosciences).

Immunohistochemistry

Mice were transcardially-perfused with PBS followed by 4%paraformaldehyde (PFA, Sigma). Frozen midbrain sections (30 μm) wereimmunostained for Mac-1 (CD11b, 1:1000; Serotec). Fluorojade C staining(Millipore) was performed on adjacent sections according tomanufacturer's protocol to assess degenerating neurons and quantifiedusing ImageJ. Overall dopaminergic neuron survival was assessed sevendays following MPTP intoxication and resolution of cell death processeswith polyclonal antibody to mouse Tyrosine hydroxylase (TH), 1:1000(Calbiochem) and counterstained for Niss1 substance by thionin staining(Tieu et al. (2003) J. Clin. Invest., 112:892-901) as previouslydescribed (Benner et al. (2004) Proc. Natl. Acad. Sci., 101:9435-9440).Total numbers of Mac-1⁺ cells, TH- and Niss1-stained neurons in the SNpcwere estimated by stereological analysis with StereoInvestigatorsoftware (MicroBrightfield) using the optical fractionator module(Liberatore et al. (1999) Nat. Med., 5:1403-1409). Quantitation ofstriatal TH (1:500; Calbiochem) was performed by densitometric analysisas described (Benner et al. (2004) Proc. Natl. Acad. Sci.,101:9435-9440). Adjacent midbrain sections were immunostained for CD4(L3T4; 1:200, BD Pharmingen). Sections were incubated instreptavidin-horseradish peroxidase (HRP) solution (ABC Elite vectorkit, Vector Laboratories), and color developed using a generation systemconsisting of diaminobenzidine (DAB) chromogen (Sigma-Aldrich) asdescribed (Benner et al. (2004) Proc. Natl. Acad. Sci., 101:9435-9440).

Statistical Analyses

All values are expressed as means±SEM. Differences among means wereanalyzed by one-way ANOVA followed by Fisher's least significantpost-hoc testing for multiple comparisons (SPSS, Inc.). All effects oftreatment were tested at the 95% confidence level.

Results N-4YSyn Immunity Exacerbates the MPTP-Induced NigrostriatalLesion

To test whether N-α-Syn-induced immunocytes could exacerbateMPTP-induced inflammation and dopaminergic neurodegeneration,splenocytes (SPC) from donors immunized with N-4YSyn were adoptivelytransferred to MPTP-recipients and the extent of inflammation andneurodegeneration. Stereological analysis of Mac-1+ cells within theSNpc 2 days post-MPTP showed greater than 16-fold increase in numbers ofMac-1+ cells compared with PBS controls (FIG. 35A), while adoptivetransfer of N-4YSyn SPC to MPTP-recipients exacerbated reactive Mac-1+cell numbers/mm² by 35% greater than that observed with MPTP alone and96% greater than PBS controls. Fluorojade C (FJ-C) staining of dead ordying neurons revealed that adoptive transfer of N-4YSyn SPC acceleratedMPTP-induced neuronal death by 7.2-fold (FIG. 35B). Analysis ofsurviving nigral TH⁺ neurons 7 days after MPTP intoxication indicated a45% overall neuronal loss compared with PBS controls, whereasMPTP-treated recipients receiving N-4YSyn SPC exhibited a 63% reductionof TH⁺ neurons (FIG. 35C). PBS mice that received N-4YSyn SPC showed nochange in TH⁺ neuron numbers compared to PBS controls. MPTP mice thatreceived SPC from PBS/adjuvant- or non-nitrated α-syn(4YSyn)/adjuvant-immunized donors showed no significant additive orprotective effect on microglial activation or neuronal survival comparedto MPTP alone as previously described. No significant effects of anytreatment were observed among numbers of non-dopaminergic neurons(TH-Niss1+). Analysis for CD4+ T cell infiltration revealed thatMPTP-intoxicated recipients of N-4YSyn SPC had increased infiltration ofCD4+cells within the SNpc following adoptive transfers, whereasMPTP-intoxication alone showed limited infiltrates at 48 hour-postintoxication and no CD4+ cells were identified in PBS-treated controls(FIG. 35D). These data demonstrate that adaptive immune responsesagainst N-α-syn exacerbate MPTP-induced neuroinflammation andnigrostriatal degeneration and support the previous works above.

T cells isolated from N-4YSyn donors stimulated in vitro with anti-CD3for 24 hours produced greater concentrations of IL-17a and TNF-αrelative to naïve T cells (FIG. 35E). N-4YSyn antigenic stimulation ofCD4+ effector T cells isolated from immunized mice induced theproduction of IL-17a, TNF-α, IFN-γ and IL-2, but not IL-4 or IL-5 (FIG.35F) indicating that immunization partially polarized the CD4+ T cellsin vivo towards either a Th1 or Th17 phenotype. Functionalcharacterization of Treg isolated from immunized FoxP3-GFP mice revealedthat Treg were functionally deficient in the capacity to inhibiteffector T cell proliferation to anti-CD3 stimulation followingimmunization with N-4YSyn (20%) as compared to Treg isolated from naïvedonors (80%) at a ratio of 1:1 (FIG. 35G).

N-4YSyn Immunity Toxic Effect is Mediated by CD4+ T Cell Subsets

In order to identify the CD4+ T cell subset responsible for the toxiceffects mediated by N-4YSyn immunity, CD4+ T cells were isolated fromN-4YSyn immunized donors and polarized in vitro for 5 days in cultureconditions to favor either a Th1, Th2 or Th17 phenotype and thenadoptively transferred into MPTP-intoxicated recipients as shown (FIG.36A). CD4+ T cells not adoptively transferred were re-stimulated for 4hours with PMA and ionomycin then supernatants collected for analysis ofcytokine production after 24 hours. Cytometric bead array analysisconfirmed that the CD4+ T cell subtypes were indeed polarized to thedesignated phenotype characterized by IFN-γ production by Th1 cells,IL-4 by Th2 cells, and IL-17a by Th17 cells (FIG. 36B).

Analysis of TH immunostained ventral midbrain and striatum 7 days afterMPTP treatment and adoptive transfers revealed that adoptive transfer ofboth N-4YSyn Th1 and Th17 subsets resulted in decreased numbers ofsurviving TH+ neurons within the SNpc; whereas, only N-4YSyn Th17 cellsresulted in diminished TH termini density within the striatum (FIG.36C). Stereological analysis of ventral midbrain sections indicated thatwhile PBS-treated controls averaged 7994±212 total TH+ neurons withinthe SNpc, MPTP-intoxication induced a 25% loss of TH+ neurons to5971±250. Adoptive transfer of N-4YSyn Th1 cells increased the lesioninduced with MPTP by 28% resulting in 4320±252 total TH+ neurons;whereas, adoptive transfer of N-4YSyn Th2 cells had no significantadditive or exacerbative effect on the total TH+ neurons in response toMPTP-intoxication. In contrast, adoptive transfer of N-4YSyn Th17 cellsinduced a 53% decrease in the number of surviving TH+ neurons (2800±243)relative to MPTP-intoxication alone reaching statistical significancerelative to all other treatment groups (FIG. 36D). Analysis of THdensity within the striatum showed that TH density within the striatumof MPTP-intoxicated mice was 26% relative to PBS-treated controls.Adoptive transfer of neither N-4YSyn Th1 nor Th2 cells did notsignificantly affect the striatal density relative to MPTP-intoxicationalone. In contrast, adoptive transfer of N-4YSyn Th17 cellssignificantly exacerbated the MPTP-induced loss of striatal TH densityto 5% of PBS-treated controls (FIG. 36E).

Co-Transfer of VIP SPC with N-4YSyn SPC Attenuates N-4YSyn MediatedMicroglial Responses

As VIP increases Treg numbers or suppressive function (Delgado et al.(2005) J. Leukoc. Biol., 78:1327-1338; Gonzalez-Rey et al. (2006) Blood107:3632-3638) or through abrogation of Th17 differentiation (Leceta etal. (2007) Neuroimmunomodulation 14:134-138), it was determined whetherVIP modulation of N-α-syn-directed immune responses could affectneurodegenerative activities. SPC populations from VIP-treated orN-4YSyn-immunized mice were adoptively transferred either separately ortogether to MPTP-recipients and the neuroinflammatory andneurodegenerative responses were evaluated. MPTP mice that receivedN-4YSyn SPC exhibited an exacerbated nigral Mac-1 response, which wasdiminished in mice treated with VIP SPC (FIG. 37A). Similarly, FJ-Cstaining was increased in N-4YSyn SPC-treated MPTP mice; whereas, theFJ-C staining in mice treated with pooled VIP and N-4YSyn SPC wasdiminished. In validation of these observations in MPTP-intoxicatedmice, transfer of VIP SPC reduced the numbers of activated microglia by33%; whereas, transfer of N-4YSyn SPC numbers increased Mac-1+ densitiesby 35% (FIG. 37B). Importantly, co-transfer of VIP SPC with N-4YSyn SPCnot only attenuated the exacerbative effects mediated by N-4YSyn SPC by75%, but diminished numbers of Mac-1+ microglia 39% less than MPTPalone. Analysis of nigral Mac-1+ microglia 7 days post-MPTP demonstratedsustained microglial activation in both mice that received N-4YSyn SPCalone or in combination with naïve SPC; whereas, significant numbers ofMac-1+ cells were not observed within any other treatment group.Analysis of total nigral FJ-C⁺ cells revealed a 7-fold increase inMPTP-recipients that received N-4YSyn SPC compared with MPTP alone;while 36% fewer neurons were injured or dead after transfer of combinedVIP- and N-4YSyn-SPC (FIG. 37C). Adoptive transfer of naïve SPCs aloneor together with N-4YSyn SPC showed no significant detrimental orprotective effect on microglial activation, and numbers of FJ-C⁺ neuronswere not significantly different compared with MPTP alone. These dataindicated that a cell population within the VIP, but not naïve SPC, wasbetter able to inhibit or suppress N-4YSyn mediated effector cells andabrogate neuropathology.

VIP SPC Modulate N-4YSyn Immunity to Confer Neuroprotection

To validate that neuroprotection was not a transient effect, THimmunostained ventral midbrain and striatal sections were assessed 7days after MPTP treatment and adoptive transfers. MPTP mice thatreceived VIP SPC showed a modest increase in TH+ neuronal density. Incontrast, TH+ neurons within the SNpc of N-4YSyn SPC recipients werediminished compared with MPTP alone; whereas, those that received pooledVIP and N-4YSyn SPC exhibited TH⁺ neuronal densities reminiscent of PBScontrols (FIG. 38A). Although lesions of the dopaminergic striataltermini are typically more severe, similar patterns of dopaminergic lossare observed in mice treated with MPTP alone and with separate SPCpopulations, while those treated with N-4YSyn SPC and VIP SPC exhibiteddiscernable increases in the density of TH⁺ striatal termini.Stereological analysis and comparison with PBS controls showed that MPTPreduced SNpc TH⁺ neurons by 48%, while MPTP and N-4YSyn SPC reducedthose neurons by 64% (FIG. 38B). MPTP recipients of VIP SPC showed amodest, yet significant 18% increase in TH⁺ neuron number compared toMPTP alone. In comparison, 91% of TH⁺ neurons survived in mice receivingSPC from N-4YSyn-immunized and VIP-treated donors. Striatal TH⁺ densityin MPTP-intoxicated mice was 16% of PBS controls, whereas transfer ofN-4YSyn SPC to MPTP recipients reduced densities to 7% of PBS controls(FIG. 38C). Although transfer of VIP SPC to MPTP-mice showed nosignificant additive or protective, co-transfer of VIP and N-4YSyn SPCincreased striatal termini survival to 39% of PBS controls. Adoptivetransfer of naïve SPC alone showed no significant detrimental orprotective effect on dopaminergic neuronal survival.

To elucidate the neuroprotective cell populations within the SPC pools,Treg from naïve and VIP-treated donors were enriched and each populationwas adoptively transferred with N-4YSyn SPC into MPTP mice. Co-transferof N-4YSyn and VIP SPC to MPTP-recipients provided 100% protection ofTH⁺ nigral dopaminergic neurons; whereas, significant protection was notobserved in MPTP mice that received SPC from N-4YSyn-immunized and naïvedonors (45% TH⁺ neuron survival) compared with percentages of survivingneurons after treatment with MPTP alone (49%) or in combination withN-4YSyn SPC (34%) (FIG. 38D). In comparison, adoptive transfer of Tregfrom either naïve or VIP-treated donors with N-4YSyn SPC affordedsignificant protection with VIP-Treg being more effective (96% survival)than naïve Treg (80% survival). Analysis striatal dopaminergic terminiwere comparable, showing that N-4YSyn SPC exacerbated the MPTP-inducedlesion to 13% of PBS controls (FIG. 38E). Co-transfer of naïve withN-4YSyn SPC was not effective in perturbing N-4YSyn exacerbativeeffects; whereas co-transfer of VIP and N-4Syn SPC increased survival to47% of PBS controls, as did co-transfer of naïve Treg and N-4YSyn (50%of PBS controls). Co-transfer of Treg from VIP-treated mice with N-4YSynSPC was the most efficacious increasing the mean terminal density to 62%of PBS controls. These results demonstrate that VIP-Treg can protectagainst N-4YSyn adaptive immune degenerative activities.

Immunization with N-4YSyn or treatment with VIP altered the frequenciesof splenic CD3, CD19+, CD4+, and CD4+ CD25+ cells (FIG. 39A). Flowcytometric analysis for CD4+ CD25+T cells within SPC populationsrevealed that VIP-treated donors had increased percentages of CD4+CD25+T cells with greater than 95% of this population also being FoxP3⁺.Analysis of antigen-induced proliferative responses showed that whileN-4YSyn T cells proliferated in response to N-4YSyn, naïve orVIP-treated donors did not. In contrast, anti-CD3 stimulation of T cellsfrom all donor groups induced proliferative responses in excess of10-fold. Such responses, however, were not observed against non-nitratedα-syn in any of the experimental or control groups.

To assess whether VIP SPC suppress effector T cell proliferativeresponses, SPC co-cultures from N-4YSyn immunized and VIP treated donorswere evaluated for their proliferative capacity in the presence ofeither anti-CD3 or N-4YSyn. At a one-to-one ratio of N-4YSyn SPC to VIPSPC, proliferation to both anti-CD3 stimulation and N-4YSyn weresuppressed by 67% and 81%, respectively and diminished in a dosedependent fashion with the diminution of VIP SPC number. Given thedichotomy between Treg and Th17 differentiation, it was hypothesizedthat Treg function or development may be inhibited by immunization withN-4YSyn. To test this hypothesis, CD4+ CD25+CD62L^(low) Treg isolatedfrom naïve, N-4YSyn-immunized, and VIP-treated mice were evaluated fortheir capacity to inhibit CD3-mediated proliferation of CD4+ CD25-naïveT cells (FIG. 39B). VIP-Treg were increased in their functional capacityto suppress T cell proliferation compared with naïve Treg showing a 5%greater inhibition of proliferation. Importantly, N-4YSyn-Treg werefunctionally deficient in their suppressive function of Teffproliferation showing 3-fold less percent inhibition of Teffproliferation compared to naïve Treg. In contrast, pooled VIP- andN-4YSyn-Treg showed enhanced suppressive capacity compared to all otherTreg populations, with 10% greater inhibition versus naïve Treg at aone-to-one responder:Treg ratio. These data indicate that restoration offunctional Treg with VIP abrogates N-4YSyn-immunity.

To characterize effector and regulatory T cell subsets fromN-4YSyn-immunized or VIP-treated mice, isolated CD4+ T cells separatelyor pooled at a 1:1 ratio were stimulated to induce cytokine expression.Quantitative RT-PCR revealed that N-4YSyn T cells showed increasedexpression of Th17 and Th17-associated genes relative to naïve T cells.This included interleukin (IL)-21, IL-17A and transcription activatorRAR-related orphan receptor c (Rorc), whereas genes linked to Th1[signal transducer and activator of transcription (Stat)4, IL-6, andinterferon (Ifn)-γ], Th2 [Gata3, Stat6, IL-4, IL-10, and IL-13], andTreg [forkhead box P3 (Foxp3) and IL-10] (Kaiko et al., 2008) weredecreased (FIG. 39C). The increased expression of IL-17 and Rorc withconcomitant decrease in Th1, Th2, and Treg associated genes, suggestedthat N-4YSyn immunization polarized CD4+ T cells toward a Th17phenotype. Moreover, genes encoding cytokines known to inhibit Th17differentiation including IL-2, IL-4, IL-15, and IFN-γ were decreased inN-4YSyn T cells compared with naïve T cells. Interestingly, VIP T cellsshowed few changes in gene expression relative to naïve T cells, withpredominately genes associated with a Th1 phenotype decreased inexpression whereas expression of Th2, Treg, or Th17 related genes werenot affected. In comparison, pooled N-4YSyn- and VIP-T cells showeddecreased expression of Th1 and Th17 related genes; whereas, genes forTreg were increased (FIG. 39C).

Analysis of cytokine production in response to anti-CD3 stimulationshowed increased production of IL-17A and IL-6 from N-4YSyn T cellsrelative to naïve T cells; while, production of IL-2, IFN-γ (FIG. 39D),and IL-4 were decreased. TNF-α production was also increased greaterthan 2.5-fold relative to naïve T cells. Analysis of cytokine productionby VIP-T cells revealed that the cytokine production was notsignificantly different to that of naïve T cells, although production ofTh2-related cytokines IL-10 (FIG. 39D), IL-4, and IL-13 (data not shown)were marginally increased. In comparison, co-culture of N-4YSyn andVIP-T cells resulted in increased production of regulatory cytokinesincluding IL-10, IFN-γ (FIG. 39D), and IL-13, with concomitant decreasedproduction of IL-17a and IL-6 compared with N-4YSyn T cells. TGF-β1production was increased 2-fold relative to naïve T cells insupernatants of N-4YSyn-immunized, VIP-treated, and pooled T cellpopulations.

It was next theorized that VIP could induce antigenic tolerance whengiven with N-4YSyn immunization. To test this idea, T cells from N-4YSynimmunized donors treated with or without VIP were assessed forproliferation capacity to N-4YSyn antigen. T cell proliferation toN-4YSyn was suppressed 2.5-fold in T cells isolated from N-4YSyn and VIPimmunized donors compared to N-4YSyn alone (FIGS. 39E and F). These dataindicate that VIP and N-4YSyn immunization induce antigen-specifictolerance to N-4YSyn. Moreover, T cells from N-4YSyn-immunized andVIP-treated mice showed a 2-fold increase in expression of genesencoding Foxp3 and IL-10. This demonstrated that VIP favors theinduction of Treg responses (FIG. 39G). Possible mechanisms couldinclude divergent transcriptional regulation either in favor ofRORγt/RORc or FoxP3 pathways, as induction of one activator oftranscription may inhibit the induction of the other (Awasthi et al.(2008) J. Clin. Immunol., 28:660-670; Kom et al. (2008) Proc. Natl.Acad. Sci., 105:18460-18465).

Example 7

A spectrum of neurological dysfunctions is associated with advancedHIV-1 infection and termed HIV-1-associated neurocognitive disorders(HAND) (Antinori et al. (2007) Neurology 69:1789-1799). In the era ofantiretroviral therapy and increased patient survival, nervous systemimpairment is more subtle with low level infection and focalneuroinflammation more closely correlated with mild neuropsychologicalsigns and symptoms. The pathological correlate of HAND is encephalitis.Before the widespread use of antiretroviral drugs, encephalitis wascharacterized by the presence of multinucleated giant cells, profoundviral replication, astrogliosis, microgliosis, myelin pallor, andneuronal dropout with severe compromise of dendritic arbor (Hult et al.(2008) Int. Rev. Psychiatry., 20: 3-13; Chemer et al. (2002) Neurology59:1563-1567; Masliah, E. (1996) Am. J. Pathol. 149:745-750). HIVencephalitis (HIVE) remains prevalent, although attenuated by effectivedrug treatments. Patients show significant CNS lymphocytic infiltratesas a consequence of disease or immune reconstitution syndrome. Moreover,HIVE depends on the continuous flux of activated leukocytes toward thebrain parenchyma rather than simply autonomous HIV infection andinflammation per se. CD4+ T cells as well as CD8+ T lymphocytesaccumulate in brains of patients with progressive HIV-1 infection andAIDS (Gisslen et al. (2007) J. Neuroimmune Pharmacol., 2:112-119; Yilmazet al. (2008) J. Acquir. Immune. Defic. Syndr., 47:168-173; Spudich etal. (2005) BMC Infect. Dis., 5:98). Prior work examined T lymphocytesubsets in the CA1, CA3, and CA4 regions of the hippocampus of AIDSpatients with and without HIVE and showed that hippocampalactivated/memory CD45RO+T lymphocytes were significantly increased indiseased hippocampal subregions (Petito et al. (2003) J. Neurovirol.,9:36-44). This led to the notion that perineuronal location of CD4+cells provides the potential for lymphocyte-mediated neuronal injury ortrans-receptor-mediated neuronal infection (Petito et al. (2003) J.Neurovirol., 9:36-44).

The presence of activated microglia and brain macrophages with lowerlevels of virus remains a central pathological feature of disease.Increased inflammation can occur as a consequence of secreted viral andcellular proteins from activated or infected mononuclear phagocytes (MP;perivascular brain macrophages and microglia) (Persidsky et al. (2003)J. Leukocyte Biol., 74:691-701) and include proinflammatory cytokines,chemokines, and arachidonic acid and its metabolites NO, quinolinicacid, and glutamate as well as HIV-1 proteins such as Tat, Nef, andgp120 (Smith et al. (2001) J. Neurovirol., 7:56-60; Kaul et al. (2005)Cell Death Differ. 12(Suppl. 1):878-892; Kaul et al. (2005) Neurotox.Res., 8:167-186; Rostasy et al. (2005) J. Neurol. Neurosurg. Psychiatry76:960-964; Rostasy, K. M. (2005) Neuropediatrics 36:230239; Kadiu etal. (2005) Neurotox., Res. 8:25-50).

Host immune surveillance against persistent viral infection includesCD8+ CTL and humoral and innate secretory responses (Rosenberg et al.(1997) Science 278:1447-1450; Petito et al. (2006) J. Neurovirol.,12:272-283; Poluektova et al. (2004) J. Immunol., 172:7610-7617;Poluektova et al. (2002) J. Immunol., 168:3941-3949). However, themechanisms by which the virus escapes clearance remain unknown. Includedin these responses are attempts to purge the infected host of latentlyinfected cells (Rosenberg et al. (1997) Science 278:1447-1450; Petito etal. (2006) J. Neurovirol., 12:272-283). Nonetheless, of all immuneresponses, CD8+ T cells are among the most effective and were previouslyinvestigated in prior reports in rodent models of neuroAIDS (Poluektovaet al. (2004) J. Immunol., 172:7610-7617; Poluektova et al. (2002) J.Immunol., 168:3941-3949; Gorantla et al. (2007) J. Immunol.,179:4345-4356). It is posited herein that in addition to CTL, CD4+ CD25+regulatory T cells (Treg) as well as effector T cells (Teff) play animportant role in HAND control. Treg, a subset of CD4+ T cells, are nowwell recognized for their immune modulatory function and play pivotalroles in maintaining immunological tolerance. Their principal role is toattenuate T cellmediated immunity and suppress autoreactive T cells(Curiel et al. (2004) Nat. Med. 10:942-949; Wang et al. (2004) Immunity.20:107-118; Sakaguchi, S. (2004) Annu. Rev. Immunol., 22:531-562). Teffpromote inflammatory responses and speed recognition and immunity(Eggena et al. (2005) J. Immunol., 174:4407-4414). It is now reportedthat Treg modulate immune responses in the brain and lead to neuronalprotection in murine HIVE. Neuroprotection was found to be mediated byattenuating HIV-1-induced microglia activation and enhancing ofneurotrophic factors. These results indicate the importance of Treg inthe control of HIV-1-associated neurodegeneration in the antiretroviralera and when adaptive immune responses remain operative.

Materials and Methods Animals, Infection of Bone Marrow-DerivedMacrophages (BMM), and Induction of HIVE

Four- to 6-wk-old male C57BL/6J mice (The Jackson Laboratory) weremaintained in accordance with guidelines for the care of laboratoryanimals from the National Institutes of Health and with approval of theInstitutional Animal Care and Use Committee of the University ofNebraska Medical Center (Omaha, Nebr.). BMM were derived after a 7-dayculture of bone marrow cells with macrophage CSF (M-CSF; Wyeth) and wereinfected as previously described (Gorantla et al. (2007) J. Immunol.,179:4345-4356). Briefly, vesicular stomatitis virus (VSV)-pseudotypedHIV-1_(Yu2) (HIV-1/VSV) was used to infect BMM at a concentration of 1pg of HIV-1 p24 per cell for 24 hours. After a continuous 5-dayculture, >90% of BMM were virus positive according to HIV-1 p24immunochemical tests (Dako-Cytomation). Reverse transcriptase activityas a function of [³H]deoxythymidine triphosphate from BMM culturesupernatants confirmed the extent of infection as previously described(Gendelman et al. (1994) Adv. Neuroimmunol., 4:189-193). To induce HIVE,HIV-1/VSV-infected BMM (1×10⁶ cells/5 μl/mouse) were delivered byintracerebral (i.c.) injection into the basal ganglia of 4-wk-oldC57BL/6J mice using stereotactic coordinates as previously described(Persidsky et al. (1996) Am. J. Pathol., 149:1027-1053). Mice injectedi.c. with PBS served as sham-injected controls. Isolation, activation,and transfer of Treg and Teff From pooled splenic and lymph node CD3⁺CD4+ T cells enriched from negative selection columns (R&D System),Treg-enriched CD4+ CD25+ and naive CD4+ CD25− T cells were prepared bypositive and negative selection for CD25+ T cells, respectively, usingPE-anti-CD25 (BD Pharmingen) magnetic beads conjugated to anti-PE mAband passage over autoMACS columns (Miltenyi Biotech) as previouslydescribed (Reynolds et al. (2007) J. Leukocyte Biol., 82:1083-1094). Byflow cytometric analyses, T cells were shown to be >95% enriched foreach T cell subset. Isolated CD4+ CD25+Treg and CD4+ CD25− T cells wereactivated by culture in the presence of 0.5 μg/ml anti-CD3 (145-2C11; BDPharmingen) and 100 U/ml mouse rIL-2 (R&D Systems). Three days later,1.0×10⁶ activated Treg or Teff (anti-CD3 stimulated CD4+ CD25− T cells)were harvested and adoptively transferred i.v. to HIVE mice.

BMM and Treg/Teff Cocultivations

BMM were seeded at 1×10⁶/well in 6-well plates containing a 1:1 ratiomixture of BMM and T cell medium. BMM and HIV-1/VSV-infected BMM werecocultivated with Treg or Teff for 6 days. Supernatants were collectedas conditioned medium (CM). BMM viability was measured using theLIVE/DEAD viability cytotoxicity kit (Invitrogen) after removal of thecocultured Treg and Teff. Cell viability was measured by MTT assay (Douet al. (2007) Virology 358:148-158).

Measures of Oxidative Stress

To assess hydrogen peroxide (H₂O₂) production from uninfected orinfected BMM, cells were plated at 1×10⁵/0.2 ml tissue culturemedium/well in a 96-well fluorometer plate and stimulated for 24 hourswith 200 ng/ml mouse rTNF-α (R&D Systems) as previously described(Reynolds et al. (2007) J. Leukocyte Biol., 82:1083-1094). The mediumwas removed and replaced with Krebs-Ringer buffer (Sigma-Aldrich)containing 10 μM PMA, 0.1 U/ml HRP, and 50 μM Amplex Red(Sigma-Aldrich). BMM cultured in the absence of TNF-α or PMA served asbaseline controls. Fluorescence intensity was measured at 563 nm(excitation)/587 nm (emission) 90 minutes after the addition of AmplexRed using a microplate spectrophotometer (μQuant; BioTek Instruments)interfaced with analysis software (KC Junior; BioTek Instruments).

Isolation and Characterization of Primary Mouse Neurons

Eighteen-day-old embryonic fetuses were harvested from terminallyanesthetized pregnant C57BL/6J mice. Cerebral cortices were dissectedand digested using 0.25% trypsin (Invitrogen). Cortical digests wereseeded at a density of 1.5×10⁵ cells/well in 24-well plates containingpoly-D-lysine-coated cover slips and cultured in neurobasal mediumsupplemented with 2% B27, 1% penicillin/streptomycin, 0.2% FBS, and 0.5mM L-glutamine (Invitrogen). After 10-14 days, neuron-enriched culturescontained >90% microtubule-associated protein (MAP)-2-positive cellswith <2% glial fibrillary acidic protein (GFAP)-positive cells asdetermined by immunocytochemistry. Mature neurons were treated with CMcollected from 24-hour cocultures of HIV-1/VSV-infected BMM in thepresence or absence of Teff or Treg.

Immunohistochemistry

Brain tissues were derived from perfused mice and processed aspreviously described (Gorantla et al. (2007) J. Immunol.,179:4345-4356). Murine microglia were detected with rabbit polyclonalAbs to Iba1 (ionizing calcium-binding adaptor molecule 1) (1/500; Wako)or Mac-1 (CD11b; 1/500; Serotec). Astrocytes were visualized withantirabbit GFAP Ab (1/1,000; DakoCytomation). Anti-HIV-1 p24 Abs (1/10;DakoCytomation) were used to identify HIV-1-infected cells. PutativeTreg were identified by dual staining with anti-CD4 (1/100;DakoCytomation) and anti-Forkhead box P3 (FoxP3) (1/100; ProMabBiotechnologies) Abs (Reynolds et al. (2007) J. Leukocyte Biol.,82:1083-1094). Abs to neuronal nuclei protein (NeuN) (1/100) and MAP-2(1/1,000; Chemicon) were used to identify neurons, and mousecross-reactive chicken anti-human brain-derived neurotrophic factor(BDNF) and antiglial cell line derived neurotrophic factor (GDNF) (1/50;Promega) were used for growth factor expression. Primary Abs werevisualized with Alexa Fluor 488 (green)- and Alexa Fluor 594(red)-conjugated secondary Abs (Invitrogen; Molecular Probes). Imageswere obtained by an Optronics digital camera fixed to Nikon Eclipse E800(Nikon Instruments) using MagnaFire 2.0 software (Optronics).Fluorescence intensity in the stained area of serial brain sectionsencompassing the i.c. injection sites was analyzed under ×400magnification using NIH Image J software. To detect apoptotic neurons invitro and infected BMM in brain sections, a Roche Applied Sciences insitu cell death detection kit with alkaline peroxidase was usedaccording to the manufacturer's instructions to stain for TUNEL-positiveneurons and 4′,6′-diamidino-2-phenylindole (DAPI) as a nuclear stain.Laser-scanning images were obtained using a Nikon sweptfield laserconfocal microscope with a ×200 power field (Nikon Instruments). Aminimum of 10 images were taken from each brain section obtained frominfected controls and groups treated by adoptive transfer of Treg orTeff. The total TUNEL-positive cells and DAPI nuclei staining in eachfield were counted and the percentage of apoptotic neurons wasdetermined from the ratio of the number of TUNEL-positive cells to thetotal number of DAPI-positive cells.

Western Blot Assays

Twenty μg of protein harvested from brain or cell lysates was separatedon 10-20% Tris-Tricine gels and blotted onto polyvinylidene fluoridemembranes (Bio-Rad Laboratories). Membranes were probed overnight at 4°C. with primary Abs including rabbit polyclonal anti-caspase-3 (1/1000;Cell Signaling), rabbit polyclonal anti-GFAP (1/1000; DakoCytomation),rabbit polyclonal anti-Iba1 (1/500; Wako), chicken monoclonal anti-humanBDNF, and biotin-conjugated anti-TNF-α. Primary Abs and β-actin weredetected with HRP-conjugated goat anti-mouse (1/10,000), goatanti-rabbit (1/10,000), goat anti-chicken (1/10,000), and mouseanti-α-actin mAb (1/10,000, Sigma-Aldrich). Proteins were visualizedwith an ECL kit (Bio-Rad Laboratories).

Cytokine Arrays

Equal volumes of cell culture supernatants were incubated with theprecoated cytokine Ab array according to the manufacturer's instructions(AAM-CYT-3-2; RayBiotech). Densitometric analysis of the array wasperformed using the NIH Image J software.

Statistical Analyses

The results were expressed as mean±SEM for each group. Statisticalsignificance between groups was analyzed by Student's t test usingMicrosoft Excel. Differences were considered statistically significantat p<0.05.

Results HIVE Mice

HIVE was established using BMM infected with HIV-lNVSVpseudotyped virusand injected i.c. into the basal ganglia of syngeneic C57BL/6J mice(FIG. 40). This led to the induction of HIV-1 induced focal encephalitisalong the injection track as shown by HIV-1 immunostained cells, robustastrogliosis and microgliosis, and T cell infiltrate as evidenced bypositive staining for expression of HIV-1 p24, GFAP, Iba1, and CD3.

Treg and Teff Modulate Neural Responses in HIVE Mice

Treg have been shown to have a potential role in modulating the immuneresponse to HIV infection (Oswald-Richter et al. (2004) PLoS Biol.,2:E198; Kinter et al. (2004) J. Exp. Med., 200:331-343). In an effort toassess the role of Treg in a mouse model of HIVE, Treg and Teff T cellsubsets from naive mice were isolated and characterized. Flow cytometricanalyses indicated that Tregs were >85% CD4+ CD25+FoxP3+ and naive Teffwere >95% CD4+ CD25-FoxP3− T cells (FIG. 41A). Three days after CD3activation, >95% of CD4+ Teff showed CD25 up-regulation withoutconcomitant FoxP3 expression. Additionally, mRNA levels for FoxP3,TGF-β, and IL-10 from Treg were significantly elevated over those fromTeff, whereas expression of IL-2 and IFN-γ mRNA levels was diminished byactivated Treg and increased in Teff (FIG. 41B). Treg suppressed theproliferative response of CD3-activated Teff in a dose-dependent fashion(FIG. 41C). Taken together, the T cells used in these studies showedappropriate Treg and Teff phenotypes.

To evaluate the roles of Treg and Teff in regulating neuroinflammatoryresponses in HIVE mice, 1×10⁶ anti-CD3-activated Treg or Teff wereadoptively transferred to HIV-1/VSV-infected recipients 24 hours afterinduction of HIVE. By 7 days postinfection, immunohistochemistrystaining of tissues surrounding the injection tracks indicated thatHIV-1/VSV or HIV-1/VSV/Teff-injected mice exhibited dense GFAP and Iba1expression compared with PBS-sham controls (FIG. 41D). In contrast, bothGFAP and Iba1 expression were reduced in HIV-1/VSV/Treg-injected mice.Quantitative measurement of GFAP and Iba1 intensities confirmedsignificant increases in expression by HIV-1/VSV- andHIV-1/VSV/Teff-treated mice compared with PBS controls and significantreductions in the HIV-1/VSV/Treg group compared with the HIV-1/VSV andHIV-1/VSV/Teff groups (FIG. 41E). Of notable importance was thesignificant reduction of HIV-1 p24 levels HIVE mice treated with Tregcompared with HIV-1/VSV- and HIV-1/VSV/Teff-treated mice (FIGS. 41D and41E). Based on the observations that Treg attenuate theneuroinflammatory responses following HIV-1 infection, the ingress ofCD4+ T cells into the brain was evaluated. The presence of CD4+ cellswere observed within the injection site of mice from all treatmentgroups (FIG. 41D). CD4+ cells were significantly increased in theHIV-1/NSV and HIV-1/VSV/Teff-treated groups (FIG. 41E); however, incontrast, the ingress of CD4+ cells was diminished to the levels of shamcontrol in infected mice treated with Treg (FIGS. 41D and 41E).Interestingly, CD4+ FoxP3+ double-positive cells were present in onlythe HIV-1/VSV/Treg-treated group.

Of the microglial secretory factors known to influence secondaryneuronal degeneration, TNF-α is implicated in affecting neuronal cellloss (Hult et al. (2008) Int. Rev. Psychiatry., 20:3-13; Rostasy et al.(2005) J. Neurol. Neurosurg. Psychiatry 76:960-964; Rostasy, K. M.(2005) Neuropediatrics 36:230-239; Kitaoka et al. (2006) Invest.Opthalmol. Vis. Sci. 47:1448-1457; Nakazawa et al. (2006) J. Neurosci.26:12633-12641). Western blot analysis of brain lysates revealed thatthe expression of TNF-α was increased in HIV-1/VSV and HIV-1/VSV/Teffmice compared with sham control, whereas in HIV-1/VSV/Treg mice, TNF-αlevels were decreased to PBS sham control levels (FIGS. 41F and 41G).Similarly, levels of Iba1 and GFAP in HIV-1/VSV and HIV-1/VSV/Teff micewere increased above sham control levels, whereas in HIV-1/VSV/Treg miceIba1 and GFAP levels were diminished. These data indicate that Treg, butnot Teff, are capable of attenuating HIV-1/VSV-induced glia activationto a neuroinflammatory phenotype.

Treg-Mediated Neuroprotection in HIVE Mice

To evaluate the neuroprotective abilities of T cells for HIVE, neuronaldensity was measured in diseased animals where Treg were adoptivelytransferred. To determine a mechanism for these effects, expressions ofBDNF, GDNF, MAP2, and NeuN were measured with or without T celltransfers. Evidence of neuronal dropout was observed by NeuN/MAP-2immunostaining (FIG. 42A). Densitometric analysis of neurons revealedthat HIV-1/VSV-infected mice showed 40 and 75% reductions in MAP2 andNeuN staining, respectively, and Teff-treated HIVE mice showed MAP2 andNeuN staining reductions comparable to those of HIVE mice (FIG. 42B). Incontrast, infected mice treated with Treg exhibited no significantreduction in neuronal expression of MAP2 or NeuN with neuron levelscomparable to those of sham control mice. Densitometric analysis ofcellular expression of growth factors revealed that BDNF and GDNFexpression was diminished by >40% in mice treated withHIV-1/VSV-infected BMM or those mice treated with Teff, whereas levelsof growth factor expression in infected mice treated with Treg werecomparable to or exceeded that of sham-treated controls. Enhancedexpression of BDNF in HIV-1/VSV/Treg-treated mice was confirmed byWestern blot analysis (FIGS. 42C and 42D). These data, taken together,indicate that Treg enhance neurotrophin secretion and protect neurons inHIVE mice.

Treg Induces Cytotoxicity in HIV-1/VSV-Infected BMM

To elucidate mechanisms for Treg-induced neuroprotection, the effects ofTreg on HIV-1/VSV-infected BMM was investigated. It was initiallyevaluated whether Treg affected cell death of infected BMM. In theseexperiments, BMM were infected with HIV-1/VSV for 24 hours and Teff orTreg were added at a BMM:T cell ratio of 3:1. Cell viability wasdetermined by the MTT assay after 72 hours of treatment with T cellsubsets and was normalized as the percentage of uninfected BMM controlcultures. Compared with uninfected BMM, viabilities of infected BMM inthe absence of T cells or presence of Teff were diminished by 15 and20%, respectively (FIG. 43A). Most interestingly, the viability ofinfected BMM treated with Treg was reduced by 37% of uninfected BMMcontrols and diminished by >20% compared with either of the otherinfected BMM groups. These results were confirmed by LIVE/DEAD(Invitrogen) cytotoxicity staining, which demonstrated that infected BMMcultured in the absence or presence of Teff increased cytotoxicity to14.6%±2.7 and 19.9%±2.7%, respectively, compared with the cytotoxicityof uninfected BMM (FIG. 43B). In contrast, coculture of infected BMMwith Treg increased BMM cytotoxicity to 28.6%±3.9%, thus confirming thatthe previously recorded diminution of viable BMM was due to increasedcytotoxicity.

Next, it was assessed whether HIV-1-infected BMM cytotoxicity requirescell-cell contact between the infected cells and Treg. BMM were isolatedand infected with the HIV-1/VSV pseudotype virus. Treg andHIV-1-infected BMM were cocultured either by Transwell™ inserts or bydirect physical contact for 1-3 days without M-CSF. After 3 days, BMMwere depleted of Tregs by removing the inserts and by serial washing(3×) and were assessed for viability by MTT assay. Compared withuninfected BMM, percentage of viabilities (±SEM) for HIV-1 infected BMMcultured alone, cocultured directly with Treg, or cocultured with Tregusing Transwell™ inserts, were 117±7.6, 75.3±6.2, and 159±8.6,respectively. Compared with HIV-1-infected BMM alone, BMM viability wassignificantly (p<0.05) lower when cocultured directly with Treg thanwith Treg separated by Transwell™ inserts. Thus, the lower levels ofviability exhibited by infected BMM cocultured in direct contact withTreg compared with the viability levels of those cocultured withbarrier-separated Treg support the notion that Treg-induced apoptosis ofinfected macrophage is mediated by cell-cell contact.

Additionally, to assess the effects of Treg on HIV-1/VSV infected cellapoptosis in vivo, TUNEL staining in brain sections was assessed thatencompass the i.c. injection sites from HIVE mice treated without orwith Teff or Treg. Surprisingly, TUNEL labeling was concentrated aroundthe injection tracks (FIG. 43C). Treg-treated HIVE mice exhibited agreater density of TUNEL+BMM compared with mice HIV-1/VSV andHIV-1/VSV/Teff groups. This observation suggested that Treg-inducedapoptosis of HIV-1/VSV-infected BMM confers neuronal protection in HIVEmice.

Treg Reduce HIV-1 Replication, Reactive Oxygen Species (ROS), andCytotoxicity in BMM

To test whether Treg mediated the inhibition of HIV-1 replication inHIV-11VSV-infected BMM, BMM were infected with HIV-1/VSV for 24 hours.After viral washout, Treg or Teff were applied and cocultured for 6days. Supernatants, collected at different time points, were used for anHIV-1 reverse-transcriptase activity assay. Compared with reversetranscriptase activities in HIV-1/VSV-infected BMM, levels of progenyvirion production were significantly increased by day 1 in theHIV-1/VSV/Teff group and continued to remain higher until both levelsreached a plateau at day 4 (FIG. 44A). In contrast, levels of progenyvirion in infected BMM cultures treated with Treg (HIV-1/VSV/Treg) neverapproached those of the other two infected groups and were significantlybelow those of HIV-1/VSV infected BMM by day 3 and at times thereafter.Furthermore, the numbers of multinucleated giant cells, a hallmark ofHIV-1 infection, were significantly reduced in the HIV-1 NSV/Treg group.Also, immunostaining suggested that Treg inhibited HIV-1 p24 proteinexpression in virally infected BMM (FIG. 44B). Percentages of HIV-1p24-positive BMM indicated that coculture with Treg, but not Teff,significantly reduced the number of HIV-1-infected BMM compared withHIV-1/VSV-infected BMM controls (FIG. 44C).

Because oxidative stress is known to enhance neurotoxicity by increasedlevels of superoxide radicals and NO (Reynolds et al. (2007) J.Leukocyte Biol., 82:1083-1094; Reynolds et al. (2007) Int. Rev.Neurobiol. 82:297-325), it was evaluated whether the extent that Tregmay affect ROS production as a mechanism of neuroprotective activity. Itwas hypothesized that Treg also suppress virally infected BMM-inducedtoxicity through suppression of ROS production. To test this, ROSproduction was assessed in HIV-1/VSV-infected BMM cocultured for 24hours in the absence or presence of anti-CD3-activated Teff or Treg.Compared with uninfected BMM controls, HIV-1NSV-infected BMM resulted ina 4.7-fold increase in H2O2 production; however, Treg treatment ofHIV-1/VSV-infected BMM significantly decreased H₂O₂ production(p<0.001), although not to baseline control levels (FIG. 45A). Incontrast, Teff treatment of HIV-1/VSV-infected BMM failed tosignificantly affect H₂O₂ production. To test the effect of Treg on theROS responses, uninfected BMM were activated for 24 hours with PMA andTNF-α and cocultured in the absence or presence of Teff or Treg.Similarly as in infected BMM, coculture with Treg significantlysuppressed the ROS response of activated uninfected BMM, whereas Teffyielded no significant effects on ROS responses (FIG. 45B).Additionally, cytokine secretion was analyzed by a membrane-basedcytokine array. Array analysis showed increased expression of IL-2,IL-12, MCP-1, and MCP5, by HIV/VSV-infected BMM or infected BMM treatedwith Teff compared with uninfected BMM (FIGS. 45C and 45D). In contrast,treatment of infected BMM with Treg diminished IL-2, IL-12, MCP-1, andMCP5 to levels below those attained either after infection or afterinfection and culture in the presence of Teff.

Treg Induce Neuroprotective Responses from HIV/VSV-Infected BMM CM

To substantiate the protective capacity of Treg to attenuate neuronaltoxicity, neuronal cell death was measutred in primary neuronal culturescultured for 24 hours in the presence or absence of CM from uninfectedBMM (control) or HIV-1/VSV-infected BMM cultured in the absence orpresence of Teff or Treg. Expression of MAP-2 and NeuN by primaryneurons confirmed the neuronal integrity of control CM-treated neurons.TUNEL staining showed more apoptotic neurons in cultures after treatmentwith CM from HIV-1/VSV BMM and HIV-1 VSV/Teff BMM compared with controlCM, whereas treatment with CM from HIV-1/VSV/Treg BMM showed fewerTUNEL-positive neurons (FIG. 46A). Quantitation of apoptotic neuronsconfirmed that the percentages of apoptotic neurons were significantlyincreased after treatment of primary neurons with CM from HIV-1/VSV BMMand HIV-1/VSV/Teff BMM compared with control CM (FIG. 46B). In contrast,treatment of neurons with CM from HIV-1/VSV/Treg BMM significantlydiminished percentages of apoptotic neurons to levels attained withcontrol CM.

Example 8

Innate immune dysfunction is a pathogenic feature of amyotrophic lateralsclerosis (ALS) (Boillee et al. (2006) Neuron 52: 39-59; Lobsiger et al.(2007) Nat. Neurosci., 10:1355-1360). Transgenic (Tg) miceoverexpressing mutated human G93A superoxide dismutase 1 (SOD 1) (Rosenet al. (1993) Nature 362:59-62) recapitulate ALS pathobiology includingneuroinflammatory responses and motor neuron degeneration (Gurney et al.(1994) Science 264:1772-1775; Hall et al. (1998) Glia 23:249-256; McGeeret al. (2002) Muscle Nerve 26:459-470; Turner et al. (2004)

Neurobiol. Dis., 15:601-609). Microglial inflammatory responsescontribute to progressive neuronal loss in SOD1 mutant Tg mice and inhuman ALS (Beers et al.

(2006) Proc. Natl. Acad. Sci., 103:16021-16026; Boillee et al. (2006)Science 312:1389-1392; Clement et al. (2003) Science 302:113-117; Mardenet al. (2007) J. Clin. Invest., 117:2913-2919; Wu et al. (2006) ProcNatl Acad. Sci., 103:12132-12137). Functional ties between adaptiveimmunity and neurodegenerative disease are known for Parkinson's disease(Baba et al. (2005) Parkinsonism Relat Disord., 11:493-498; Bas et al.(2001) J. Neuroimmunol., 113:146-152), Alzheimer's disease (AD) (Casalet al. (2003) Clin Biochem., 36:553-556; Scali et al. (2002) NeurobiolAging 23:523-530; Shalit et al. (1995) Clin Immunol Immunopathol.,75:246-250), and multiple sclerosis (MS) (Bar-Or et al. (2003) Brain126:2738-2749; Filion et al. (2003) Clin Exp Immunol., 131:324-334).Moreover, neuroprotective responses by Copolymer-1 (COP-1) immunizationwere observed in animal models of these and other neurodegenerativedisorders (Aharoni et al. (2005) Proc Natl Acad. Sci., 102:19045-19050;Avidan et al. (2004) Eur J. Immunol., 34:3434-3445; Bakalash et al.(2005) J Mol. Med., 83:904-916; Benner et al. (2004) Proc Natl Acad.Sci., 101:9435-9440; Butovsky et al. (2006) Proc Natl Acad. Sci.,103:11784-11789; Gorantla et al. (2007) J. Immunol., 179:4345-4356;Gorantla et al. (2008) Glia 56:223-232; Kipnis et al. (2000) Proc NatlAcad. Sci., 97:7446-7451; Laurie et al. (2007) J. Neuroimmunol.,183:60-68; Liu et al. (2007) Eur. J. Immunol., 37:3143-3154; Schori etal. (2001) J. Neuroimmunol., 119:199-204). However, links betweenadaptive immunity and ALS remains obscure. Changes in T cell numbers andadaptive immune molecules in postmortem ALS and SOD1Tg mouse nervoussystem tissues were reported (McGeer et al. (2002) Muscle Nerve26:459-470; Alexianu et al. (2001) Neurology 57:1282-1289; Graves et al.(2004) Neuron Disord., 5:213-219; Henkel et al. (2006) Mol CellNeurosci., 31:427-437; Henkel et al. (2004) Ann Neurol., 55:221-235;Kawamata et al. (1992) Am J. Pathol., 140:691-707; Troost et al. (1990)Neuropathol Appl Neurobiol 16:401-410). Interestingly, such COP-1immunization strategies yielded mixed results in G93A-SOD1 mice (Angelovet al. (2003) Proc Natl Acad. Sci., 100:4790-4795; Habisch et al. (2007)Exp Neurol., 206:288-295; Haenggeli et al. (2007) Neurobiol Dis.,26:146-152). Taken together, these findings suggest a progressive immunedysfunction in G93A-SOD1 mice.

Mutant SOD1 may play a role in progression of ALS as microglia recoveredfrom G93A-SOD1 mice induce increased motoneuron injury than microgliafrom over-expressing wild-type (Wt) human SOD1 (Beers et al. (2006)Proc. Natl. Acad. Sci., 103:16021-16026; Xiao et al. (2007) J.Neurochem., 102:2008-2019). Human ALS immunocytes show that bothactivated monocytes and T cell numbers are linked to disease progression(Zhang et al. (2005) J. Neuroimmunol., 159:215-224; Zhang et al. (2006)J. Neuroimmunol., 179:87-93). These data are consistent with a diseasemodel where systemic immunologic activation plays an active role in ALSprogression (Alexianu et al. (2001) Neurology 57:1282-1289; Zhang et al.(2005) J. Neuroimmunol., 159:215-224; Xiao et al. (2007) J. Neurochem.,102:2008-2019; Zhao et al. (2006) J. Neurochem., 99:1176-1187).

Based on these observations, T cell phenotype and function in G93A-SOD1Tg mice and in ALS patients was investigated. COP-1 immunizationprovided clinical benefit to only female G93A-SOD1 Tg mice. Profound Tcell functional deficits were observed in pre-symptomatic male G93A-SOD1 Tg mice spleen as well as acute lymphopenia in end stage animals.Transfer of naive lymphoid cells from Wt donor mice to SOD1 Tg recipientmice failed to affect survival or overcome the observed lymphopenia. AsCOP-1 is linked to neuroprotective T regulatory cells (Treg) and themodulation of neuroinflammatory responses (Benner et al. (2004) ProcNatl Acad. Sci., 101:9435-9440; Laurie et al. (2007) J. Neuroimmunol.,183:60-68), it was then investigated whether CD4+ CD25+Treg or CD4+CD25− T effector cells (Teff) could affect neurological deficits andsurvival. Importantly, for SOD1 Tg mice, polyclonal-activated Wt Treg orTeff administered by adoptive transfer extended longevity and attenuatedmotor deficits. Treg delayed clinical symptom onset, while Teffincreased latency from onset to late stage disease. These resultstogether with supportive data in human ALS indicate the presence ofaberrant T cell subsets in disease.

Materials and Methods Animals

Mice from two SOD1 Tg mouse strains expressing the G93A mutation,B6SJL-TgN(SOD1*G93A)1Gur (stock number, 002726; hereafter designatedB6SJL SOD1 Tg) and B6.Cg-Tg (SOD1*G93A)1Gur/J (stock number, 004435;hereafter designated B6 SOD1 Tg), and age- and sex-matched Wtlittermates were obtained from Jackson Laboratory (Bar Harbor, Me.).B6SJL SOD1 Tg mice survive from 16-20 weeks, while B6 SOD1 Tg mice havea delayed survival phenotype of 19-22 weeks. Mice were randomlyseparated to control and treatment groups upon receipt. All animalprocedures met with National Institutes of Health guidelines and wereapproved by the Institutional Animal Care and Use Committee (IACUC) ofthe University of Nebraska Medical Center.

Human Subjects

Experimental procedures involving human subjects were conducted inconformance with the policies and principles contained in the FederalPolicy for the Protection of Human Subjects (U.S. Office of Science andTechnology Policy) and in the Declaration of Helsinki.

COP-1 Immunization

B6 SOD1 Tg mice (7 weeks old) were immunized with 75 μg of COP-1 in 0.1ml PBS weekly (q1wk) or every 2 weeks (q2wk), or treated with PBS alone.Subcutaneous injections were administered in the flanks with a 50 μlbolus given to each side.

Spleen Morphology, Weight, Viable Cell Counts

Spleens from Wt and Tg mice were measured and weighed. Single cellsuspensions were prepared by pressing spleens through 60 μm sterile wiremesh screens in Hanks' balanced salt solution (HBSS, Mediatech Inc.,Herndon, Va.). Erythrocytes were lysed with ammonium chloride potassiumbuffer and leukocytes washed by centrifugation. Numbers of viablesplenic leukocytes were determined by trypan blue exclusion ofhemocytometer counts.

Lymphocyte Proliferation

Splenocytes from individual animals were plated in 96-well round-bottomplates at 16106 cell/ml in RPMI medium 1640 (Gibco, Carlsbad, Calif.)supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 25 mM HEPES,1 mM sodium pyruvate, 1× nonessential amino acids, 55 mM2-mercaptoethanol, 100 units/ml penicillin, and 100 μg/ml streptomycin(complete RPMI 1640) (Mediatech Inc.). Quadruplicate replicates werestimulated with anti-CD3 (1 μg/ml) (clone 145-2C11, BD Pharmingen), goatanti-IgM (20 μg/ml) (Jackson Immuno Research, West Grove, Pa.) orcultured in media alone at 37° C. in 5% CO₂ for 3 days. From Tg miceimmunized with COP-1 (Sigma-Aldrich, St. Louis, Mo.), spleen cells werecultured in the presence of COP-1 (5 μg/ml), concanavalin A (Con A, 2μg/ml, Sigma-Aldrich), or media for 5 days. Cells were pulsed for thefinal 18 hours of incubation with 1 μCi [³H] methylated thymidine([³H]-TdR) (MP Biomedicals ICN, Solon, Ohio), harvested onto glass-fiberfilters, and counted by β-scintillation spectrometry (Top Count, PackardInstrument Co., Meriden, Conn.). Levels of spleen cell proliferation foreach animal were normalized to levels of proliferation obtained fromcells cultured in media alone and were reported as a stimulation index.

Immunohistochemical Assays

Fresh frozen spleens of Tg mice and Wt littermates were embedded in OCTmedia (Sakura Fintek, Torrance, Calif.) and sectioned at 10 μm using acryostat (CM1900, Leica, Bannockburn, Ill.). Sections were collected onslides and fixed in ice-cold acetone-methanol (1:1) for 30 minutes.Slides were washed in phosphate-buffered saline (PBS) at roomtemperature (RT) and quenched for endogenous peroxidase activity in 3%hydrogen peroxide in methanol for 15 minutes. Nonspecific staining wasblocked with 5% normal rabbit serum (NRS) (Vector Laboratories,Burlingame, Calif.) in PBS for 1 hour. For immunostaining, primaryantibodies (clone designations and dilutions) included anti-CD3 (clone17A2, 1:100), anti-CD19 (clone 1D3, 1:100), anti-F4/80 (clone BM8,1:500) and anti-Gr-1 (clone RB6-8C5, 1:100) (all obtained fromeBioscience, San Diego, Calif.). Sections were incubated with primaryantibody diluted in PBS/5% NRS for 90 minutes at room temperature,washed in PBS and incubated with polyclonal rabbitanti-rat:immunoglobulin (1:400) (Dako, Capinteria, Calif.) for 30minutes followed by streptavidin-horseradish peroxidase solution (ABCElite vector kit, Vector Laboratories) for 30 minutes. Staining wasvisualized by addition of hydrogen peroxide substrate anddiaminobenzidine chromogen (DAB substrate kit for peroxidase, VectorLaboratories) solution. Sections were counterstained with hematoxylin(Surgipath Medical Industries, Inc., Richmond, Ill.), dehydrated,covered with mounting media (Cytoseal 60, Kalamazoo, Mich.) and mountedwith a glass coverslip. Slides were examined under a light microscope(Eclipse E800, Nikon, Inc., Melville, N.Y.) and representative imagescaptured at 100× magnification. Follicle counts, area per follicle, anddensities of CD3, CDl9, F4/80 and Gr-1 expression were evaluated from 4fields/animal by digital image analysis using Image-Pro Plus version 4software (Media Cybernetics, Silver Spring, Md.).

Flow Cytometric (FCM) Analysis of Mouse and Human Leukocytes

Single cell suspensions of spleens from Wt and Tg mice were stained withfluorescein isothiocyanate (FITC)-conjugated (clone designate) anti-CD19(1D3), anti-CD4 (RM4-4), anti-CD62L (MeI-14), and anti-Gr-1 (RB6-8C5);phycoerythrin (PE)-conjugated (clone) anti-CD4 (GK1.5), anti-CD8b(53-5.8), and F4/80 (BM8, eBioscience); and allophycocyanin(APC)-conjugated (clone) anti-CD3 (145-2C11) and anti-CD44 (1M7). Allantibodies except where indicated were obtained from BD Pharmingen (SanDiego, Calif.).

Peripheral blood from 10 ALS patients and their age-matched caregiverswere collected in ethylenediaminetetraacetic acid (EDTA) containingglass tubes at Columbia University, shipped overnight, and processedupon arrival at the University Nebraska Medical Center. Complete bloodcount (CBC) and differential analysis for each donor and patient weredetermined from samples obtained prior to shipping. For FCM analysis, 20μl of appropriate fluorochrome-conjugated antibodies were added to 100μl of whole blood and incubated in the dark for 30 minutes at roomtemperature. Erythrocytes were lysed and leukocytes fixed with FACSLysing solution (BD Biosciences). Antibodies (clone) utilized in thesestudies included FITC-conjugated anti-CD8a (RPA-T8), anti-CD16 (55661),anti-CD45RA (HI1100), and anti-CD19 (H1B19); PE-conjugated anti-CD14(55715) and anti-CD4 (OKT4); and APC-conjugated anti-CD3 (UCHT1),anti-HLA-DR (LN3), and anti-CD45R0 (UCHL1).

Stained mouse and human leukocytes were evaluated by FCM analysis usinga FACSCalibur flow cytometer interfaced with CellQuest software(BD-Biosciences, Immunocytometry Systems). Electronic bit maps wereutilized to encompass and gate lymphocyte and monocyte subsets duringFCM analysis.

Measures of Lymphocyte Apoptosis and Necrosis

Spleen cells from 14 weeks old Wt and Tg mice were evaluated as freshisolates or were stimulated for 24 or 48 hours as for lymphocyteproliferation. Harvested spleen cells were stained with annexin-V-FITC(ApoptosisDetection kit, Calbiochem/EMD Biosciences, Inc., San Diego,Calif.), PE-anti-Thy-1 (clone 53-2.1) to detect T cells, and APCanti-CD45R/B220 (clone RA3-6B2) to detect B cells (eBioscience).Actinomycin D (7-ADD; BD Pharmingen) was used as a viable exclusionindicator for membrane permeability to distinguish apoptotic(annexin-V+7-ADD-) from necrotic cells (annexin-V+7-ADD+), the latterhaving lost membrane integrity.

Isolation and Purification of CD4+ CD25+(Treg) and CD4+ CD25-(Teff)Cells

Treg and Teff cells were isolated as previously described. Lymph nodes(cervical, mandibular, axillary, brachial, inguinal and mesenteric) andspleens were harvested from male Wt B6 mice (9 weeks old). After lysisof red blood cells, T cell populations were enriched by negativeselection on CD3+ T cell columns (R&D Systems, Minneapolis, Minn.). CD3+T cells were further passed through CD4+ T cell subset enrichment column(R&D Systems) to obtain a highly pure CD4+ T cell population in theeluted fraction. The CD4+ T cell fraction was incubated with PE-labeledanti-CD25 antibody (BD Pharmingen) followed by anti-PE microbeads(Miltenyi Biotec, Auburn, Calif.) and subjected to magnetic separation(Auto MACS, Miltenyi Biotec). Nonadherent cells were eluted from themagnetic column and were enriched for CD4+ CD25− Teff cells, whileadherent cells eluted from the column were enriched as CD4+ CD25+Tregcells. Purity of nonadherent and adherent cell fractions were determinedby FCM analysis (FACSCalibur flow cytometer, BD Biosciences) usingantibodies that recognize disparate epitopes to CD3, CD19, CD4, CD8,CD25, and Foxp3 (eBioscience). Prior to activation, fresh isolates ofTregs were >95% CD4+ CD25+Foxp3+ while Teff were >95% CD4+ CD25-Foxp3-.To activate and expand enriched T cell populations, purified cells werecultured for 4 days in 24-well plates at 1×10⁶ cells per ml of completeRPMI 1640 with 0.5 μg/ml anti-CD3 (145-2C11; BD Pharmingen) and 3×10⁶irradiated splenocytes (3,300 rads). CD4+ CD25+T cells required theaddition of 100 U/ml of mouse recombinant interleukin (IL)-2 (R&DSystems). Furthermore, CD4+ CD25+Tregs exhibited increased expression ofmRNA for Foxp3, TGF-β and IL-10, whereas Teff showed increasedexpression of IFN-α mRNA. Tregs also inhibited anti-CD3 inducedmitogenesis in a dose-dependent manner.

Adoptive Cell Transfers

Freshly isolated lymphocytes obtained from spleens of naive Wt B6 donormice and anti-CD3 activated Treg or Teff cells after 4 days ofstimulation in vitro were harvested, washed, and resuspended in HBSS. ToB6 SOD1 Tg recipient mice, 850×10⁶ lymphocytes or 1×10⁶ Treg or Teffcells in 0.25 ml of HBSS or PBS alone were administered intravenousevery 6 weeks at 7, 13, and 19 weeks of age.

Body Weight and Clinical Signs

The initial sign of the disease is a high frequency resting tremor thatprogresses to gait impairment, asymmetrical or symmetrical paralysis ofthe hind limbs, followed by complete paralysis at end stage. Beginningat 7 weeks of age, all animals were assessed weekly for body weight andfor signs of motor deficit with the following 4 point-scoring system: 4points if normal (no sign of motor dysfunction), 3 points if hind limbtremors were evident when suspended by the tail, 2 points if gaitabnormalities were present, 1 point for dragging of at least one hindlimb, and 0 point for symmetrical paralysis (Weydt et al. (2003)Neuroreport., 14:1051-1054). Disease onset was determined at theearliest presentation of symptoms (i.e. score=3). Mice that reached aclinical score of 0 or lost 20% of maximum body weight were deemedunable to survive, removed from the study, immediately euthanized, andscored as a terminal event.

Paw Grip Endurance (PaGE) Test

Grip strength of hind limbs of mice were assessed each week aspreviously described (Weydt et al. (2003) Neuroreport., 14:1051-1054).Each mouse was placed on the wire-lid of a conventional housing cage andgently shaken to prompt the mouse to hold on to the grid. The lid wasturned upside down and the duration determined until the mouse releasedboth hind limbs. Each mouse was given three attempts with a maximumduration of 90 seconds and the longest latency was recorded.

Rotarod Performance

Mice were pre-conditioned for 3 days prior to testing then monitored forrotarod performance once every week starting at 7 weeks of age(Haenggeli et al. (2007) Neurobiol Dis 26:146-152). In brief, mice wereplaced on a partitioned rotating rod (Rotamex Rota-rod apparatus,Columbus Instruments, Columbus, Ohio) and tested at a 5, 10, and 15 rpmfor a maximum of 90 sec at each speed with a minimum of 5 minutes restbetween attempts. The overall rotarod performance (ORP) was calculatedas the area under the curve using Prism (version 4, Graphpad GraphpadSoftware, San Diego, Calif.) from the plot of the time that the animalremained on the rod as a function of the rotation speed.

Statistical Analyses

All values are expressed as mean±SEM. Differences among normallydistributed means were evaluated by Student's t test for two groupcomparisons or one-way ANOVA followed by Bonferroni or Fisher's LSDpost-hoc tests for pairwise comparisons amongst multiple data setsexhibiting equal variances or by Dunnett's post-hoc tests for dataexhibiting unequal variances (Statistica v7, StatSoft, Tulsa, Okla., andSPSS v13, SPSS, Inc., Chicago, Ill.). Cox's F-test comparison wasperformed for comparison between treatment groups for Kaplan-Meieranalyses.

Results COP-1 Immunization of B6 SOD1 Tg Mice

The initial works investigated whether COP-1 immunization of B6 SOD1 Tgmice affect disease progression. In these experiments male and female B6SOD1 Tg mice were immunized with 75 μg COP-1 s.c. in 0.1 ml PBS eitherevery week (q1wk) or every other week (q2wk), or animals were treatedevery week with PBS as excipient controls. Kaplan-Meier analysisindicated that weekly COP-1 immunization had an affect on the lifespanof SOD1 Tg mice compared to PBS controls (p=0.0413), howeverimmunization every other week did not increase survival (p=0.1673) (FIG.47A). For mice immunized weekly with COP-1, the mean age of survivalincreased by 9.9% compared to PBS controls (p=0.006), while COP-1immunization every other week increased the mean age of survival by6.1%, however this did not reach significance. Log-normal analysis ofmortality probability at 10 day intervals showed that COP-1 immunizationevery week and every other week initially provided protective benefitscompared to PBS controls; however, by 160 days of age, the probabilityof mortality for mice immunized every other week evolved to thatafforded by PBS controls (FIG. 47B). Kaplan-Meier analysis of treatedmice stratified for gender indicated that increased survival by weeklyimmunization was associated with female mice (p=0.0434), but had noeffect on survival of male Tg mice (FIG. 47C). Similarly, compared toPBS treated controls, immunization with COP-1 every week increased themean age of survival for female, but not male Tg mice, and immunizationevery other week produced no difference in mean age of survival foreither male or female Tg mice (FIG. 47D). These results posed thequestion as to whether adaptive immunity was fully functional inpre-symptomatic SOD1 Tg mice.

Impaired T Cell Immune Responses in SOD1 Tg Mice

Based on the failure of the COP-1 immunization strategies to increaselongevity in male SOD1 Tg mice, and preliminary data showing diminishedspleen size and immune responses with age, it was tested whether T cellresponses were functional. These studies revealed that T cell immunefunction elicited in B6 SOD1 Tg male mice was significantly impaired by19 weeks of age. Spleen cells from 4 and 8 week-immune SOD1 Tg mice,stimulated in vitro with COP-1 exhibited increased stimulation indicescompared to those cultured in media alone (dashed line), whereas cellsfrom PBS treated mice were unable to respond to COP-1 (FIG. 47E)indicating that immunization strategies elicited functional COP-1responsive T cells in early stage of the disease. However, after 12weeks of weekly or bi-weekly immunizations, stimulation indices of COP-1stimulated spleen cells diminished to levels statistically indiscerniblefrom those of cells cultured in media alone indicating that the T cellimmune responses in those mice had waned. In concomitant assays to testthe overall functionality of all T cell populations, spleen cellcultures were stimulated with Con A, a T cell mitogen. Stimulationindices of Con A induced T cells from B6 SOD1Tg mice in all treatmentgroups after 4 and 8 weeks (at 11 and 15 weeks of age) weresignificantly above those of media control cells (dashed line) (FIG.47F), demonstrating the presence of functional T cells in those mice.However in Tg mice at 19 weeks of age, after 12 weeks of treatment,stimulation indices of Con A stimulated T cells were indistinguishablefrom those cultured in media alone. Regression analysis of stimulationindices of Con A stimulated T cells from PBS controls indicated aprogressively diminished proliferative capacity of T cells that wasstrongly associated with increasing age of B6 SOD1Tg mice (r²=0.6308,p=0.002). Taken together these results suggest a global dysregulation ofT cell function with age in SOD1 Tg mice.

Spleen Size, Weight and Cell Counts in SOD1 Tg Mice

Based on marginal protection achieved by COP-1 immunization andprogressively diminished T cell function with age, the adaptive immunesystem was investigated in disease whereby spleens from B6SJL G93A-SOD1Tg mice were compared at early symptomatic stage (14 weeks of age) andend stage (20-22 weeks of age) with those of age and sex-matched Wtlittermate controls. All Tg mice at 14 weeks of age exhibited hind limbtremors. Morphologically, spleens from 14 weeks old B6SJL Tg mice wereidentical to those of Wt controls (FIG. 48A, left panel), whereas,spleens from end stage mice showed marked reduction in size compared tocontrols (FIG. 48A, right panel). Similarly, no differences in spleenweights from pre-symptomatic and symptomatic B6SJL mice compared to Wtwere discerned, whereas at end stage, spleen weights were diminished by45% in B6SJL Tg mice (19 weeks old) and by 59% in B6 Tg mice (22 weeksold) (FIG. 48B). No differences in gross morphology or weights fornon-lymphoid kidneys or livers were discernible between Tg and Wt miceat any age. For end stage B6 Tg mice, total viable spleen cell numberswere diminished by 70% compared to Wt controls, whereas no differencesin spleen cell numbers were observed between Tg and Wt mice in earlysymptomatic stage (FIG. 47C).

Immune Tissue Analyses of SOD1 Tg Mice

To assess splenic architecture in end stage mice, the expression of CD3,CD19, F4/80, and Gr-1 in fresh frozen sections was assessed from endstage B6 Tg mice (22 weeks old), B6SJL Tg mice (19 weeks old), andage-matched Wt controls. Splenic architecture in end stage B6 Tg andB6SJL Tg mice revealed remarkable alterations in follicle number, size,and expression of hematopoietic lineage markers compared to Wt B6littermates. Splenic follicular architecture appeared diminished with agreater number of follicles in each field for B6 SOD1 Tg (FIGS. 49B,49E, 49H, and 49K) and B6SJL SOD1 Tg (FIGS. 49C, 49F, 49I, and 49L) micecompared to Wt controls (FIGS. 49A, 49D, 49G, and 49J). The density ofCD3+ T cells in the spleen appeared unaltered in B6 Tg (FIG. 49B) orB6SJL Tg (FIG. 49C) mice compared to Wt mice (FIG. 49A), whileexpression of F/480 (FIGS. 49D, 49E, and 49F) and Gr-1 (FIGS. 49G, 49H,and 49I) in the perifollicular area of spleen appeared increased inSOD1Tg mice and the density of CD19 expression by B cells within thefollicles of SOD1 Tg mice (FIGS. 49K and 49L) appeared diminishedcompared to Wt controls (FIG. 49J). These observations were validated bydigital image analysis in B6 Wt and B6 SOD1 Tg mice. In Tg mice, splenicfollicular area was diminished by 67% (FIG. 50A) and numbers offollicles/mm² were increased by 41% (FIG. 50B) compared to Wt controls.No difference in the densities of splenic CD3 expression could beascertained between Tg and Wt control mice (FIG. 50C). In the splenicperifollicular area of Tg mice compared to Wt controls, the mean densityof F4/80 was increased by 47% (FIG. 50D), density of Gr-1 expressingcells was increased by 165% (FIG. 50E), while the intrafolliculardensity of CD19 expression was diminished by 88% (FIG. 50F).

Impaired Lymphocyte Proliferation and Necrosis in Spleens of SOD1 TgMice

Based on diminished T cell responses and observations of profoundlymphopenia in G93A-SOD1 Tg mice at end stage, splenic lymphocytephenotype and function was assessed in early symptomatic (14 weeks old)B6SJL SOD1 Tg mice to detect early immune cell aberrations in spleen.Flow cytometric analysis of CD62L and CD44 expression on CD4+ gatedsplenic lymphocytes (FIG. 51A) demonstrated a diminished percentage ofCD4+ CD44^(hi)CD62L^(lo) memory T cells and an increased percentage ofCD4+ CD44^(lo)CD62L^(hi) naive T cells compared to Wt mice whichresulted in an increased ratio of naive/memory CD4+ T cells in Tg micecompared to Wt controls (FIG. 51B). To assess lymphocytic demise,apoptotic (annexin-V+7-ADD2) and necrotic (annexin-V+7-ADD+) lymphocyteswas evaluated among viable T cells (Thy-1+) and B cells (CD45R/B220+)from spleen cell isolates of early symptomatic (14 weeks old) B6SJL SOD1Tg mice and Wt controls. Flow cytometric analysis revealed that Tg micehad a greater than 2-fold increase in the percentage of annexin-V+7-ADD+necrotic splenic T cells and a 30% increase in the percentage ofannexin-V+7-ADD− apoptotic T cells compared to Wt littermates (FIG.51C). Similarly, percentages of necrotic (41%) and apoptotic (38%) Bcells were increased in Tg mice compared to control mice.

To assess lymphoid cell function of early symptomatic B6SJL SOD1 Tg miceat 14 weeks of age, T cells were stimulated with anti-CD3 and B cellswith anti-IgM and evaluated their proliferative capacity of eachlineage. T cell proliferation in B6SJL Tg mice was significantlydiminished compared to Wt littermates; however no diminution of B cellfunction could be ascertained (FIG. 51D). The diminished T cellproliferative responses thus confirmed previous findings (FIG. 47F). Itwas also assessed whether lymphoid cells from Tg and Wt mice weredifferentially susceptible to activation-induced cell death at either 24or 48 hours post-activation, however no differences in induction ofapoptotic or necrotic T or B cells at any time point after activationwere observed.

Survival of B6 SOD1 Tg Mice after Adoptive Transfer of Naive LymphoidCells, or Activated Treg or Teff Subsets

Based on the above findings in SOD1 Tg mice demonstrating a lack ofprotective response by COP-1 immunization in male mice, diminished Tcell functional capacity in early symptomatic and late stage mice, andend stage lymphopenia, a strategy was tested to rectify the lymphoiddysregulation and extend survival by adoptive transfer of B6 Wt naivelymphoid cells to recipient B6 SOD1 Tg mice. B6 SOD1 Tg mice treatedwith 50×10⁶ naive spleen cells at 7, 13, and 19 weeks of age yielded nosignificant differences in the cumulative proportion of survival (FIG.52A, p=0.2035) or mean age of survival (FIG. 52B, p=0.315) compared toPBS-treated mice. However, mean clinical scores analyzed by factorialANOVA revealed significant improvement of reconstituted mice compared toPBS-treated controls (FIG. 52C, p=0.000001). Additionally, Kaplan-Meieranalysis showed immune reconstituted (RCS) Tg mice exhibited delayedsymptom onset (clinical score=3) (FIG. 52D, p=0.0012) as well as delayedentry into late stage (clinical score=1) (FIG. 52E, p=0.0191). However,after onset of symptoms, survival of reconstituted Tg mice trended to bediminished compared to the PBS controls as determined by Kaplan-Meieranalysis (FIG. 52F, p=0.2021) and by mean latency after onset to deathfor PBS-treated (64.5±2.6) and RCS (52.0±4.3) mice (p=0.0167).

No significant differences in body weight were discerned between RCS-and PBS-treated groups as a function of age by factorial ANOVA(p=0.5824). Additionally, no differences in PBS-treated or RCS groupswere found in the cumulative proportion (p=0.2744) and the mean age(p=0.2921) of Tg mice that reach 10% loss of maximum body weight.Although an early effect in hind grip strength was observed between10-13 weeks of age as determined by PaGE, no effects were discerniblethereafter. Factorial ANOVA indicated no differences in motor functionas determined by ORP in PBS-treated or RCS groups over their lifetime(p=0.8862).

Based on the previous results which demonstrated that regulatory T cellswere neuroprotective in a mouse model of Parkinson's disease, it wasevaluated whether activated T cell subsets also provide analogousprotection in SOD1 Tg mice. Adoptive transfer of 1×10⁶ enrichedpolyclonal-activated Wt Treg (CD4+ CD25+) or Teff (CD4+ CD25−) to B6SOD1 Tg mice at 7, 13, and 19 weeks of age led to significant increasesin longevity as determined by Kaplan-Meier analysis (FIG. 53A) and meanage of survival (FIG. 54A) compared to PBS-treated controls. FactorialANOVA of treatment and age showed that by 11-12 weeks of age, clinicalscores were increased by reconstitution of B6 Tg recipients withactivated Wt Treg or Teff compared to PBS controls (FIG. 53B,p=0.00004). Moreover, entry into late stage disease (clinical score=1)was delayed by reconstitution with Treg or Teff as determined byKaplan-Meier analysis (FIG. 54C) and mean age of entry into late stage(FIG. 54B). Of interest, transfer of activated Wt Treg, but not Teff toB6 Tg recipient mice delayed disease onset as evaluated by clinicalsigns (clinical score=3) and as determined by Kaplan-Meier analysis(FIG. 54D, p=0.002) and mean age of disease onset (FIG. 54C, p, 0.0003).On the other hand, transfer of activated Wt Teff, but not Treg to B6 Tgrecipients, increased the latency from onset (clinical score=3) to entryinto late stage (clinical score=1) as determined by Kaplan-Meieranalysis (FIG. 53E, p=0.0098). Adoptive transfer of Treg or Teffaffected weight gain and loss as determined by factorial ANOVA foreffects of treatment as a function of age (p=0.0356). Transfer ofactivated Teff, but not Treg delayed the age at which recipients lost≧10% of maximum body weight compared to PBS treatment as determined byKaplan-Meier analysis of proportion (FIG. 53F, p=0.003) and mean age(FIG. 54D) of mice reaching ≧10% weight loss.

Adoptive transfer of either Treg or Teff to B6 SOD1 Tg mice alsoimproved motor function compared to PBS treated controls as determinedby factorial ANOVA for effect of treatment with age on ORP (p=3.361028)and PaGE (p=6.7610211). Activated Wt Treg or Teff delayed loss ofrotarod performance as determined by Kaplan-Meier analysis of theproportion (FIG. 55A) and mean age (FIG. 55B) of mice at which ≧75% ofORP was reduced. Also transfer of activated Treg or Teff delayed theinitial loss of ORP compared to PBS controls as determined by thecumulative percentage (FIG. 55C) and the mean age (FIG. 55D) of micethat reach ≧25% loss of ORP. Hind limb strength was also assessed byPaGE. Compared to PBS controls, adoptive transfer of activated Treg orTeff delayed the loss of hind limb strength as determined byKaplan-Meier analysis of the cumulative percentage (FIG. 55E) andincreased mean age (FIG. 55F) of SOD1 Tg mice exhibiting ≧75% reductionof PaGE. In addition, transfer of activated Wt Treg or Teff to Tg micedelayed early loss of hind limb grip strength compared to controls asdetermined by Kaplan-Meier analysis of the cumulative percentage (FIG.55G) and mean age (FIG. 55H) of mice that exhibit ≧25% reduction ofPaGE. Of interest, after one round of Treg and Teff adoptive transfer at49 days of age, Teff appear less efficient than Treg to attenuate earlygrip loss in Tg mice, however after a second round (at 91 days of age),the capacities to attenuate loss of grip strength by Teff and Treg werecomparable (FIG. 55G).

Preliminary Studies of Altered Adaptive Immunity in ALS Patients

To assess immune alterations in ALS patients, peripheral bloodmononuclear cells (PBMC) from 10 ALS patients and 10 age-matchedcaregivers were characterized. Peripheral blood counts indicated asmall, though insignificant increase in the number of leukocytes inpatients compared to caregivers. ALS patients exhibited an increase inthe mean percentage of polymorphonuclear neutrophils (PMNs)(8.0±0.07×10⁹/L compared with 6.6±0.06×10⁹/L, p=0.022), with slightlyreduced levels of lymphocytes (20.7%±2.4% compared with 25.9%±1.8,p=0.054), and no discernible differences in monocyte levels (p=0.35).Flow cytometric analysis showed no differences in levels of peripheralblood CD19+B cells or CD3+ T cells among patients and caregivers.However compared to caregivers, ALS patients exhibited an 11.8% decreasein the percentage of CD4+ CD8− T cells (p=0.032) and a 22.9% increase inthe frequency of CD4-CD8+ T cells (p=0.043) compared to age-matchedcontrols. Additionally, the CD45RA/CD45R0 (naive/memory) ratio amongCD4+ T cells of ALS patients (0.6±0.1) was diminished by 45% compared tocaregivers (1.1±0.2, p=0.028), which was due to the diminution in levelsof CD45RA+ naive T cell among CD4+ cells (37.2%+2.5% compared with47.6%+5.1% for caregivers, p=0.0435) and a concomitant increase inlevels of CD45R0+ memory cells among CD4+ T cells (62.1%±2.6% comparedwith 51.7%±5.2% for caregivers, p=0.0465).

Example 9

A randomized, double-blind, sham-controlled trial will be performed. Atotal of 40 study participants are enrolled in the trial. These will bematched in age and sex and tempo of disease progression. Neurologicalstatus will be mild to moderate impairment with all participantsphysically independent and the ability to ambulate without assistance.Twenty participants will receive an injection of 2 μg of the C-terminaltail peptide of soluble oxidized alpha synuclein given by intradermalinjection with and without 2 μg of nanoformulated vasoactive intestinalpeptide to maximize dendritic cell responses. The other 20 participantswill receive sterile saline solution. Repeat boosting injections will beadministered every two weeks for six weeks. Study participants will beassessed for treatment effects by standardized Parkinson's diseaseratings including clinical and neurological parameters for movement,gain, and coordination. The primary endpoint for the study will be aclinical assessment of motor function at 6 months using the UnifiedParkinson's Disease Rating Scale (UPDRS). All participants in the studywill also be monitored for safety for 12 months following theimmunization procedure. If the primary endpoint is met following theanalysis of 6 month data, then the sham control participants will beoffered the opportunity to crossover into an open label study of theNeuropel immunization therapy, if they continue to meet all entrycriteria.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A method of treating a central nervous system disease or disorder ina patient in need thereof, wherein said central nervous system diseaseor disorder is characterized by the presence of at least one abnormalprotein, said method comprising administering to said patient: a) atleast one immunogen capable of inducing a humoral immune responseagainst said abnormal protein, and b) at least one adjuvant thatstimulates regulatory T cells.
 2. The method of claim 1, wherein saidcentral nervous system disease or disorder is selected from a groupconsisting of Parkinson's Disease, Alzheimer's Disease, amyotrophiclateral sclerosis, neuroAIDS, Chron's Disease, and Huntington's Disease.3. The method of claim 1, wherein said adjuvant is selected from thegroup consisting of vasoactive intestinal peptide, vitamin D,granulocyte macrophages colony stimulating factor, and transforminggrowth factor beta.
 4. The method of claim 3, wherein said adjuvant isvasoactive intestinal peptide.
 5. The method of claim 1, wherein saidcentral nervous disease or disorder is Parkinson's Disease and saidimmunogen is nitrated alpha synuclein.
 6. The method of claim 1, whereinsaid central nervous disease or disorder is Parkinson's Disease and saidimmunogen is a fragment of nitrated alpha synuclein.
 7. The method ofclaim 1, wherein said central nervous disease or disorder is Alzheimer'sDisease and said immunogen is amyloid beta.
 8. The method of claim 1wherein said central nervous system disease or disorder is amyotrophiclateral sclerosis and said immunogen is superoxide dismutase.
 9. Themethod of claim 1, wherein said immunogen and said adjuvant are in asingle composition, wherein said composition, optionally, furthercomprises at least one pharmaceutically acceptable carrier.
 10. Themethod of claim 1, wherein said immunogen and said adjuvant are inseparate compositions, wherein each composition, optionally, furthercomprises at least one pharmaceutically acceptable carrier.
 11. Themethod of claim 10, wherein said separate compositions are administeredsimultaneously.
 12. The method of claim 10, wherein said separatecompositions are administered sequentially.
 13. A compositioncomprising: a) at least one immunogen capable of inducing a humoralimmune response against at least one abnormal protein of a centralnervous system disease or disorder, and b) at least one adjuvant thatstimulates regulatory T cells.
 14. The composition of claim 13, furthercomprising at least one pharmaceutically acceptable carrier.
 15. Thecomposition of claim 13, wherein said adjuvant is selected from thegroup consisting of vasoactive intestinal peptide, vitamin D,granulocyte macrophages colony stimulating factor, and transforminggrowth factor beta.
 16. The composition of claim 13, wherein saidimmunogen is selected from the group consisting of nitrated alphasynuclein, amyloid beta, and superoxide dismutase.